Vascular injury of immature epiphyses impair stem cell engraftment in cartilage defects

Abstract

The purpose of our study was to investigate if vascular injury in immature epiphyses affects cartilage repair outcomes of matrix-associated stem cell implants (MASI). Porcine bone marrow mesenchymal stromal stem cells (BMSCs) suspended in a fibrin glue scaffold were implanted into 24 full-thickness cartilage defects (5 mm ø) of the bilateral distal femur of six Göttingen minipigs (n = 12 defects in 6 knee joints of 3 immature pigs; age 3.5-4 months; n = 12 defects in 6 knee joints of 3 mature control pigs; age, 21-28 months). All pigs underwent magnetic resonance imaging (MRI) at 2, 4, 12 (n = 24 defects), and 24 weeks (n = 12 defects). After the last imaging study, pigs were sacrificed, joints explanted and evaluated with VEGF, H&E, van Gieson, Mallory, and Safranin O stains. Results of mature and immature cartilage groups were compared using the Wilcoxon signed-rank test. Quantitative scores for subchondral edema at 2 weeks were correlated with quantitative scores for cartilage repair (MOCART score and ICRS score) at 12 weeks as well as Pineda scores at end of the study, using linear regression analysis. On serial MRIs, mature joints demonstrated progressive healing of cartilage defects while immature joints demonstrated incomplete healing and damage of the subchondral bone. The MOCART score at 12 weeks was significantly higher for mature joints (79.583 ± 7.216) compared to immature joints (30.416 ± 10.543, p = 0.002). Immature cartilage demonstrated abundant microvessels while mature cartilage did not contain microvessels. Accordingly, cartilage defects in immature joints showed a significantly higher number of disrupted microvessels, subchondral edema, and angiogenesis compared to mature cartilage. Quantitative scores for subchondral edema at 2 weeks were negatively correlated with MOCART scores (r = - 0.861) and ICRS scores (r = - 0.901) at 12 weeks and positively correlated with Pineda scores at the end of the study (r = 0.782). Injury of epiphyseal blood vessels in immature joints leads to subchondral bone defects and limits cartilage repair after MASI.

Figure 1

Sagittal SPGR 3D FS knee MRI of the immature and mature pigs after MASI. In the immature knee joint (a–c), at 2 weeks after MASI (a), MRI demonstrates MSC implants (dashed lines) in full-thickness cartilage defects (arrows). On follow-up imaging scans at week 4 (b), the knee joint demonstrates associated subchondral edema (asterisks) and bone defect in the subchondral bone (arrows). The follow-up imaging study of the immature joint at 12 weeks after MASI (c) demonstrates a persistent size of the cartilage defect and increasing size of the subchondral bone defect (arrows). In the mature knee joint (d–f), at 2 weeks after MASI (d), MSC implants (dashed lines) in full-thickness cartilage defects and intact subchondral endplate (arrows) are appreciated. In contrast to the immature joint, on follow up MR imaging of the mature joint, the knee joint demonstrates limited subchondral edema (asterisks) and the size of the cartilage defects and subchondral bone defects (arrows) decreases at week 4 (e), and week 12 (f) after MASI, consistent with progressive healing.

Figure 2

Longitudinal evaluations of cartilage defects in immature and mature cartilage. (A) Sagittal SPGR 3D FS knee MRI and macroscopic evaluation of cartilage defects in immature and mature joints at different time points after MASI. (a,b) MRI of two representative immature knee joints at 12 weeks after MASI demonstrates persistent cartilage defect which extends into the subchondral bone. (c,d) MRI of immature knee joints at 24 weeks after MASI demonstrates further increased cartilage and subchondral bone defects. (e,f) By comparison, a cartilage defect in a mature joint has been almost completely repaired at 24 weeks after MASI. There is no subchondral edema or defect. (g,h) Macroscopic specimen of the immature knee joints demonstrated incomplete cartilage defect repair at 12 weeks and increasing defects in the cartilage and (i,j) subchondral bone at 24 weeks. (k,l) By comparison, macroscopic specimens of the mature knee joints demonstrated complete cartilage defect repair at 24 weeks. (B) Corresponding Pineda cartilage score of cartilage repair in immature (after 12 and 24 weeks) and mature joints (after 24 weeks). Data are displayed as means and standard deviation of four cartilage defects per group. The Pineda score of cartilage defects of immature joints at 24 weeks was significantly higher compared to the Pineda score of mature joints. (C) Corresponding cartilage repair score of immature and mature pigs according to Wakitani cartilage repair scoring system (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 3

Correlation of quantitative scores for subchondral edema at 2 weeks after MASI with quantitative scores for cartilage repair (MOCART score, (A) and ICRS score, (B)) at 12 weeks and Pineda scores at 24 weeks (C) and correlation of the quantitative scores for Pineda scores at 24 weeks with quantitative scores for cartilage repair (MOCART score) at 12 weeks (D).

Figure 4

Histological analysis of cartilage repair in immature (A) and mature (B) pigs at 24 weeks after MASI. (A) Immature Cartilage (scale bars: 500 µm, 200 µm, and 50 µm): H&E (a–c) and van Giesson’s (d–f) staining showed reduced cartilage repair with a thin and irregular surface of the cartilage (a,b,d,e) after MASI while proper histological cartilage with dense network of vessels was observed in control samples (c,f). Significant edema, mononuclear cell infiltration (arrow, b,e), and impaired vessels (asterisk, e) were observed after MASI. The Mallory stain confirms the presence of elastin and collagen fibers in the calcified part of cartilage (g–i) and around the vessels (arrows) indicating reduced cartilage repair after MASI. Fluorescent staining (j–l) of cartilage tissue for nucleus (j, demonstrated as blue), VEGF (k, demonstrated as red), and overlay image (l; demonstrated as purple) indicate high VEGF expression and further demonstrate the endothelium of vessels within the immature cartilage. (B) Mature Cartilage (scale bars: 500 µm and 200 µm): (m) H&E staining demonstrates complete cartilage defect repair with a smooth surface (arrow); (n) complete defect repair with proper chondrocyte morphology (arrow); (o) a normal control without cartilage defect demonstrates a thin cartilage layer with smooth cartilage surface. (p) Van Gieson’s staining demonstrates complete defect repair (arrow); (q) complete defect repair with proper chondrocytes morphology (arrow); (r) a normal cartilage without defect demonstrates proper chondrocyte morphology. (s) Mallory stain demonstrates physiological collagen fiber deposition (arrow); (t) Dense network of collagen fiber distribution (arrow) and absence of vessels in cartilage; (u) a normal control cartilage with well-developed extracellular matrix. (v–x) Fluorescent staining of cartilage tissue for nucleus (v, demonstrated as blue), VEGF (w, demonstrated as red), and overlay image (x; demonstrated as purple) indicate limited VEGF expression.

Figure 5

Histological analysis of vessels in the cartilage of immature (A) and mature (B) pigs at 24 weeks after MASI, and analysis of expression of vascular endothelial growth factor (VEGF) on mRNA level using RT-PCR method (C). (A) Immature Cartilage (scale bars: 400 µm, 100 µm, and 50 µm): H&E staining revealed the presence of impaired vessels and mononuclear cell infiltration outside of the vessels (arrows, a). Alcian blue staining confirmed the presence of vessels in the immature cartilage (arrows, b). In the control samples, vessels with proper structures were observed (arrows, c). (B) Mature Cartilage (scale bars: 500 µm and 50 µm): H&E (d), and Alcian blue stainings (e) revealed no vessels in cartilage in mature cartilage after MASI (d) as well as in control pigs (f). (C) The lowest relative expression of VEGF was demonstrated in mature cartilage (Mature 24) when compared to immature (immature 12 and 24) after MASI respectively. Representative data from three independent experiments are shown ± SD (n = 3) (**p < 0.01, ***p < 0.001). SD standard deviation, ns not significant.

Figure 6

Histological analysis of cartilage repair in immature (A) and mature (B) pigs at 24 weeks after MASI (scale bars: 800 µm). Safranin O staining demonstrates the presence of microvasculature in the cartilage of immature pigs (A) while no vessels were identified in mature pigs’ cartilage (B).

Figure 7

The comparison of total cartilage repair (A), the area of repaired cartilage (um²) (B), and the percentage of impaired vessels (C) between immature and mature pigs after MASI. (A) The reduced absorbance of van Gieson dye in immature pigs was noted when compared to mature pigs. Reduced dye absorbance of van Gieson, demonstrates a limited cartilage repair and functional deterioration (***p < 0.001). (B) The reduced area of repaired cartilage (um²) in immature pigs was noted when compared to mature pigs. The lowest area of repair for cartilage defect in immature pigs at 12 and 24 months after MASI indicating on the limited cartilage repair in immature pigs (***p < 0.001). (C) Representative data from randomized, twelve fields are shown ± SD (n = 12). An asterisk (*) indicates a comparison between all of the tested groups (*p < 0.05, **p < 0.01). SD standard deviation, ns not significant.