Fibrocartilage

Samples were fixed (10% formalin, 36 h) then decalcified (10% EDTA, 6 weeks) before a dehydration and paraffin embedding were carried. Then, samples were sliced (5μm, SM2500; Leica, Nussloch, Germany) parallelly to tunnels’ longitudinal axis and fixed on glass slides (40°F oven). Standard Hematoxylin and eosin (H&E) staining was completed to evaluate graft-bone interface.
Patterns of intra-articular collagen alignment were visualized by Masson’s trichrome staining and Safranin O/fast green staining was also carried to observe fibro-cartilage formation patterns and glycosaminoglycans (GAGs) content (Chen et al., 2021 (link)).
All staining procedures were carried based on manufacturer’s instructions before an inverted light microscopy (Leica DM4000 B, Germany) was used for observation and Leica DFC420C camera (Leica Microsystems GmbH) to capture images.
Obtained results were analyzed and quantified by two observers. Three parameters (fibrocartilage formation, new bone formation and graft bonding to adjacent tissues) were considered in the final scoring (0-3 points/item, 0-9 points for total score) with higher scores representing enhanced results. Details of the scoring system are provided in Supplementary Table S2 (Cheng et al., 2016 (link)).
Immunohistochemical staining (IHC) for COL I, COL III and BMP-2 was carried. First, samples’ dewaxing and rehydration were carried before antigen-retrieval. Then, 0.3% hydrogen perioxide (20 min) and 2% bovine serum albumin (1 h) were used for blocking and primary anti-body incubation was carried over-night (4°C). Secondary antibody was used for incubation for 1 h at 37°C. Samples were then washed. Finally, observation of obtained images was completed under a light microscopy (Leica DM4000 B, Germany).

Acetabular labral samples were sliced to 3 mm thick specimens and were preserved for 2 d in 10% buffered formalin. Further, the specimens were embedded in paraffin and cut into 4 µm thick sections.
Haematoxylin and eosin staining was performed for histological analysis, and acetabular labrum degeneration was evaluated using the Krenn score for fibrocartilage degeneration in the meniscus and labrum (grades 0–3; Table 2) [17 (link),18 (link),29 (link)] by three blinded assessors who were expert hip surgeons.

En block harvesting of the patella, PLs, IFP and tibial tuberosity of the contralateral limb was performed as described above and placed in 10% NBF for 48 h. Five 5 mm tissue blocks were sharply cut perpendicular to the longitudinal fiber orientation of each ligament as illustrated in Fig. 3, from the origin at the patella/medial parapatellar fibrocartilage, to the insertion at the tibial tuberosity, taking care not to include bone and/or cartilage at the ligament origin/ insertion. Five 5 mm tissue blocks were sharply cut from the IFP, perpendicular to its longitudinal axis. Tissue blocks were embedded in paraffin and 4—6 µm sections were routinely processed with H&E staining. Tissue blocks in which metaplastic tenocytes were identified in H&E sections were also processed with toluidine blue staining. Sections were evaluated at 2.5x, 10 × and 40 × magnification by light microscopy and photographed using a Zeiss™ digital photomicroscope (AxioCam ERc5s) and then copied and stored onto a computer using designated software (Zen blue, Carl Zeiss).
All sections were assessed for diagnostic quality, and sections in which ligament structure and cellular morphology could not be assessed due to processing artefacts were omitted from the analyses. Normal ligament structure was defined as polygonal fascicles consisting of collagen bundles with scattered tenocytes, separated by endotenon recognized as interstitial connective tissue septa carrying blood vessels, lymph vessels and nerves, as illustrated in Fig. 3. Tenocytes were classified as normal when nuclei were spindle shaped (type 1) or when nuclei were plump and cigar shaped (type 2). Tenocytes with rounded nuclei situated in semi-lacunae were defined as abnormal and referred to as metaplastic tenocytes. Tissue vascularity was defined as normal when blood vessels were confined to the endotenon and abnormal when vessels were seen within fascicles.
The interfascicular endotenon was subjectively classified as thin when it was 1–2 cell layers thick, and as distinct when it consisted of multiple cell layers; the distinct interfascicular endotenon were further characterized as having a predominantly discrete or prominent vascularity as illustrated in Fig. 2. Interfascicular endotenon thickness was objectively measured on ruler-calibrated images captured at 2.5 × magnification using imaging software (ImageJ); cross-junctional areas of the endotenon were excluded from the analysis as they formed irregular structures.
Fascicle cellularity was defined as normal when scattered tenocytes were seen within the fascicles, whereas hypercellularity was defined as areas of tenocyte clustering (Fig. 2). Contrary, hypocellularity was defined as areas almost devoid of cells, which coincided with areas of metaplastic tenocytes (Fig. 2).
Ligamentous fatty infiltration was defined as normal when adipocytes were confined to the endotenon, and as abnormal when intra-fascicular adipocytes were seen. When confined to the endotenon, the amount of fatty infiltration was subjectively graded as none; slight; moderate or marked as illustrated in Fig. 2.
In the toluidine-blue sections, the extracellular matrix glycosaminoglycan content was subjectively assessed as increased in areas of increased basophilia.
Normal histological architecture of the IFP was defined as lobes of adipose tissue separated by connective tissue septa, with a synovial membrane lining along its caudal aspect and with no presence of inflammatory cell infiltration. Interlobular connective tissue septa were subjectively assessed as thin or distinct; septal thickness as well as lobular diameter were objectively measured in images acquired at 2.5 × magnification using imaging software (Image J).