Fig 2: Collagen content in cryosections from ALPPm knee joints at 6 months after injection of ALPP-labeled MSCs. (A) Representative immunofluorescence stainings for collagen type-1 (COL1, red), collagen type-II (COL2, green) and collagen type-X (COL10, red) in the cartilage defect areas. Scale bar = 100 µm. (B) Quantification of the collagen content in the defect area, 6 months post-injection. Dots represent collagen-stained area within the defect (n = 4 per group). COL1 and COL10 formed the neotissue matrix in control and serum samples, whilst IA-injection of MSCs led to enhanced COL2 and almost absent COL1 and COL10 expression in the focal cartilage lesions. *P < 0.05 by one-way ANOVA followed by Tukey’s multiple comparison test.

Fig 3: Evaluation of histochemical ALPP staining in cryosections from knees of ALPP-transgenic and WT controls, and from ALPPm knee joints with a focal cartilage defect, 1 day, 1 week, 1 month, 2 months and 6 months after injection of ALPP-labeled MSCs. (A) Representative images of histochemical ALPP staining. Insets in the upper panels show ALPP-positive staining in the synovial cavity (left), suprapatellar bursa (middle) and cartilage lesion (right), at 1 day post-injection. Most MSCs were found in the knee joint cavity. ALPP-labeled MSCs were also detected in the synovial cavity and the cartilage defect, 1 week and 1 month after MSC injection (lower panels). No MSCs from ALPP donors were detected at 2 or 6 months after ALPP-labeled MSC injection (lower panels). Scale bar = 100 µm. (B) Quantification of the ALPP-positive area in the synovium, suprapatellar bursa (sup. bursa) and defect area. Dots represent percentage of ALPP-positive area; at least 2 sections per animal were analyzed (n = 4 animals per group). *P < 0.05 vs. corresponding region 1 day after injection; #P < 0.05 vs. synovial cavity at the same time point by Kruskal-Wallis test followed by Mann-Whitney U test.

Fig 4: Toluidine Blue and Safranin O staining of cryosections from ALPPm knee joints at 1 month, 2 months and 6 months after injection of ALPP-labeled MSCs. Cartilage regeneration in the injured site was observed in the knee joints that were previously injected with 107 ALPP-labeled MSCs, suspended in rat serum, but not in ALPPm recipients injected with serum or left untreated. Scale bar = 100 µm; n = 4 per group.

Fig 5: BCIP/NBT staining of paraffin sections from various organs of ALPP-transgenic and WT controls, and of ALPPm recipient rats at 1 day, 1 week, 1 month, 2 months and 6 months after intra-articular injection of ALPP-labeled MSC. No enzyme activity could be detected in heart, spleen, kidney or liver from ALPPm recipients rats intra-articularly injected with MSC from ALPP donors. However, a few ALPP-labeled MSCs were identified in the perivascular space, surrounding pulmonary blood vessels, 1 day after injection, but only in one of the studied animals. ALPP donors show strong staining in all tissues, while no staining is observed in wild-type (WT) controls. Scale bar = 50 µm; n = 4 animals per group.