Surface markers on isolated MPCs
FACS analysis was performed (Figure 2) using cells from all four tissue samples of a single donor to exemplary define the percentage of isolated cells that expressed the surface antigens CD44, CD73, CD90 and CD105 (Table 2). All isolated cells from bone marrow, cartilage, LCF and the joint capsule were strongly positive (≥ 95 %) for CD44, CD90 and CD105 (Table 2, CD44+, CD90+, CD105+). The majority of isolated cells was also strongly positive (< 95 %) for the surface marker CD73 (Table 2, CD73+). While > 70% of cells isolated from bone marrow, cartilage and the joint capsule expressed CD73, only about half of the cells from the LCF were positve for this surface marker (Table 2, CD73+). Matching results were shown within the coexpression of CD-antigens (Figure 2; Table 2).
Histological analysis of adipogenic differentiation
In comparison to control cultures (Figure 3, b, control d 21) all cells derived from bone marrow, cartilage, the joint capsule and the LCF which were incubated in an adipogenic differentiation medium for 21 d showed positive Oil RedO stainings (Figure 3, b, differentiation d 21). The quantity of lipid droplets in all adipogenic differentiated cultures increased from d 12 (Figure 3, a, diff. d 12) to d 21 (Figure 3, a, diff. d 21). The total amount of adipocyte-like cells after 21 d was higher in culutres containing differentiated cells from the LCF and the joint capsule (Figure 3, LCF, capsule) than compared to those from bone marrow and cartilage (Figure 3, bone marrow, cartilage), as shown in representative areas.
Adipogenic marker gene expression after 21 d
After 21 d the adipogenic differentiated cultures from all four donor tissues showed a similar, clear up-regulated mean value of mRNA expression levels of adipogenic marker genes lipoproteinlipase (LPL) and peroxisome proliferator-activated receptor γ (PPARγ) in comparison to their controls (Figure 4, a). The mean expression levels of LPL in cells from LCF (Figure 4, a, LPL) and PPARγ in MPCs from the joint capsule (Figure 4, a, PPARγ) were lower than in cartilage or bone marrow-derived cells, although none of these findings were significant.
Histological and immunohistochemical analysis of osteogenesis
From d 12 (Figure 5, a, diff. d 12) to d 21 (Figure 5, a, diff. d 21) spider web-like cell patterns, surrounded by an increasing amount of extracellular, dense deposits formed in all monolayers incubated in osteogenic differentiation medium. After 21 d positive Alizarin Red S staining identified these dense depostis as calcium aggregates in all osteogenic differentiated cell-cultures from bone marrow, cartilage, the joint capsule and LCF (Figure 3, b, differentiation d 21). In addition, all osteogenic differentiated cultures showed a clearly positive, immunohistochemical staining for Col I at the end of the differentiation period after 21 d (Figure 5, c, differentiation d 21). Controls remained negative for both Alizarin Red S (Figure 5, b, control d 21) and immunohistochemical stainings (Figure 5, c, control d 21).
Osteogenic marker gene expression after 21 d
RT-PCR revealed an upregulation of the mean mRNA expression levels of osteogenic marker genes collagen type X (Col X), alkaline phosphatase (ALP) and osteocalcin (OC) in all of the osteogenic differentiated cultures compared to their controls (Figure 4, b). The expression of the osteogenic marker gene OC in MPCs derived from bone marrow was significantly higher (p < 0,05) than from MPCs isolated from cartilage (Figure 4, b, OC). Interestingly, the mean expression levels of Col I mRNA in comparison to negative controls only showed a significant increase in MPCs derived from bone marrow (Figure 4, b, Col I). Other than that, the expression of osteogenic marker genes did not differ significantly within MPCs derived from different native tissues. These findings are contrary to the clearly positive immunohistochemical stainings for Col I in all osteogenic differentiated MPCs (Figure 5, c, differentiation d 21).
Histological and immunohistochemical analysis of chondrogenesis
After 27 d of pellet-culture in presence of TGF-ß1, positive alcian blue stainings proved the presence of proteoglycans in all chondrogenic differentiated cell-pellets derived from bone marrow, cartilage, the joint capsule and LCF (Figure 6, a, differentiation d 27). In addition, chondrogenic differentiated pellets containing cells from bone marrow and cartilage showed a chondrogenic phenotype especially in outer regions of the pellets (Figure 6, a, bone marrow, cartilage). Despite clearly postive alcian blue stainings, hyaline cartilage-specific attributes, such as chondrones in areas with a low density of cells, were less visible in pellets from the joint capsule (Figure 6, a, capsule) and almost non-existent in pellets from LCF (Figure 6, a, LCF). Corresponding to alcian blue stainings immunohistochemical stainings revealed vast amounts of collagen type II (Col II) in outer regions of the differentiated pellets from bone marrow, cartilage and the joint capsule after 27 d (Figure 6, b, differentiation d 27). In comparison to the strong immunohisochemical stainings of Col II in differentiated pellets from bone marrow and cartilage (Figure 6, b, bone marrow, cartilage) the staining appeared less intense in differentiated pellets from the joint capsule (Figure 6, b, capsule). In contrast, chondrogenic differentiated pellets from LCF showed no visible stainings of Col II after 27 d (Figure 6, b, LCF). All controls remained negative for alcian blue (Figure 6, a, control d 27) and Col II (Figure 6, b, control d 27) stainings after 27 d of pellet culture in absence of TGF-ß1.
Immunohistochemical stainings for Col X were performed to examine possible chondrocyte hypertrophy during chondrogenic differentiation (Figure 6, c). While controls remained mostly negative (Figure 6, c, control d 27), differentiated pellets showed slightly stronger stainings especially in inner, cell-rich regions of the sections (Figure 6, c, differentiation d 27) that showed a less chondrogenic phenotype in earlier alcian blue and Col II stainings. The strongest matrix staining for Col X after 27 d was seen in the differentiated pellets containing cells from cartilage (Figure 6, c, cartilage).
Expression of chondrogenic marker genes after 27 d of pellet-culture
RT-PCR showed an upregulation of the mean mRNA levels of the examined chondrogenic marker genes AGG, Col II and Sox-9 in all chondrogenic differentiated pellets after 27 d (Figure 4, c). The mean mRNA expression levels of AGG and Col II were highest in chondrogenic differentiated pellets from the joint capsule although observed differences were not from statistical significance (Figure 4, c, AGG, Col II). Interestingly, the mean expression of Col II mRNA was upregulated in differentiated pellets from LCF (Figure 4, c, Col II) although the immunohistochemical stainings remained negative for Col II in corresponding pellets from LCF (Figure 6, b, LCF). Significant differences in the mean mRNA expression levels between the pellets from varying native tissues were not observed.