Response of knee fibrocartilage to joint destabilization

Abstract

Objective A major challenge to understanding osteoarthritis pathology is identifying the cellular events that precede the onset of cartilage damage. The objective of this study is to determine the effect of joint destabilization on early changes to fibrocartilage in the joint. Design/Methods The anterior cruciate ligament was transected in collagen reporter mice (Col1GFP and ColXRFP). Mineralization labels were given every two weeks to measure new mineralized cartilage apposition. Novel fluorescent histology of mineralized tissue was used to characterize the changes in fibrocartilage at 2 and 4 weeks post-injury. Results Changes in fibrocartilaginous structures of the joint occur as early as two weeks after injury and are well developed by four weeks. The alterations are seen in multiple entheses and in the medial surface of the femoral and tibial condyles. In the responding entheses, mineral apposition towards the ligament midsubstance results in thickening of the mineralize fibrocartilage. These changes are associated with increases in ColX-RFP, Col1-CFP reporter activity and alkaline phosphatase enzyme activity. Mineral apposition also occurs in the fibrocartilage of the non-articular regions of the medial condyles by 2 weeks and develops into osteophytes by 4 weeks post-injury. An unexpected observation is punctate expression of tartrate resistant acid phosphatase activity in unmineralized fibrochondrocytes adjacent to active appositional mineralization. Discussion These observations suggest that fibrocartilage activates prior to degradation of the articular cartilage. Thus clinical and histological imaging of fibrocartilage may be an earlier indicator of disease initiation and may indicate a more appropriate time to start preventative treatment.

Amplifying Bone Marrow Progenitors Expressing α‐Smooth Muscle Actin Produce Zonal Insertion Sites During Tendon‐to‐Bone Repair

“…Following euthanasia, hindlimbs were harvested and fixed in formalin for 2 days, transferred to 30% sucrose overnight, and embedded in optimal cutting temperature (OCT) compound. Tape‐stabilized, frozen mineralized sagittal sections of the knee were collected and each section was subjected to three rounds of imaging on the Zeiss Axio Scan.Z1 digital slide scanner including (i) fluorescent reporters, mineralization label, and polarized light, (ii) alkaline phosphatase (AP) fluorescent staining (Vector Blue Alkaline Phosphatase Substrate Kit; Vector Laboratories, Burlingame, CA) with Hoechst 33342 counterstain, and (iii) 0.025% toluidine blue (TB) or hematoxylin and eosin (H&E) (aqueous) staining. Sections were decalcified prior to AP staining.…”

Murine supraspinatus tendon injury model to identify the cellular origins of rotator cuff healing

“…The upper extremities were harvested and fixed in 10% formalin for 2 days at 4ºC and then placed in PBS with 30% sucrose overnight. The specimens were embedded in Cryomatrix (Thermo Scientific) and 7–8 μm tissue sections were created using a cryofilm technique [28–31]. Tissue sections were rehydrated with PBS and imaged on the Axio Scan.Z1 microscope (Zeiss) for tdTomato expression and tissue architecture via darkfield imaging.…”

High-Throughput, Multi-Image Cryohistology of Mineralized Tissues

“…This step is critical to fix the soluble cytoplasmic GFP of transgenic animals within the limits of the cell. We have utilized the cryotape for a wide range of tissue types [5][6][7][8][9][10][11][12][13] . Laboratory personnel with limited histological experience can produce high-quality sections with this method.…”

“…Recently, we have published a new high-throughput cryohistological method for assessing several response measures within a given section from mineralized tissue [5][6][7][8][9][10][11][12][13][14] . The process involves stabilizing the cryosection with frozen cryotape, adhering the taped section rigidly to a microscope slide, and conducting several rounds of staining and imaging on each section.…”