Pullulan: a new cytoadhesive for cell-mediated cartilage repair

Abstract

Introduction: Local delivery of mesenchymal stem cells (MSCs) to the acutely injured or osteoarthritic joint retards cartilage destruction. However, in the absence of assistive materials the efficiency of engraftment of MSCs to either intact or fibrillated cartilage is low and localization is further reduced by natural movement of the joint surfaces. It is hypothesised that enhanced engraftment of the delivered MSCs at the cartilage surface will increase their reparative effect and that the application of a bioadhesive to the degraded cartilage surface will provide improved cell retention. Pullulan is a structurally flexible, non-immunogenic exopolysaccharide with wet-stick adhesive properties and has previously been used for drug delivery via the wet surfaces of the buccal cavity. In this study, the adhesive character of pullulan was exploited to enhance MSC retention on the damaged cartilage surface.

Methods: MSCs labeled with PKH26 were applied to pullulan-coated osteoarthritic cartilage explants to measure cell retention. Cytocompatability was assessed by measuring the effects of prolonged exposure to the bioadhesive on MSC viability and proliferation. The surface phenotype of the cells was assessed by flow cytometry and their multipotent nature by measuring osteogenic, adipogenic and chrondrogenic differentiation. Experiments were also carried out to determine expression of the C-type lectin Dectin-2 receptor.

Results: MSCs maintained a stable phenotype following exposure to pullulan in terms of metabolic activity, proliferation, differentiation and surface antigen expression. An increase in osteogenic activity and Dectin-2 receptor expression was seen in MSCs treated with pullulan. Markedly enhanced retention of MSCs was observed in explant culture of osteoarthritic cartilage.

Conclusions: Pullulan is a biocompatible and effective cytoadhesive material for tissue engraftment of MSCs. Prolonged exposure to pullulan has no negative impact on the phenotype, viability and differentiation potential of the cells. Pullulan dramatically improves the retention of MSCs at the fibrillated surface of osteoarthritic articular cartilage. Pullulan causes an upregulation in expression of the Dectin-2 C-type lectin transmembrane complex.

Figure 1

Pullulan-conditioned cartilage. Schematic shows the methodology for conditioning cartilage with the bioadhesive pullulan prior to MSC delivery. Cartilage biopsies were taken from the articular cartilage surfaces of the tibial plateau of the knee or hip femoral head. The explant surface was coated for 10 minutes in pullulan, an α-1-4; α-1-6-glucan polysaccharide consisting of maltotriose units (A). Culture expanded PKH26-labelled MSCs were applied to conditioned cartilage explants and incubated on a rocking plate for 20 minutes. Unbound MSCs in suspension were removed by washing in PBS, leaving MSCs bound to cartilage which were fixed and analysed histologically (B). MSC, mesenchymal stem cells.

Figure 2

MSC viability and proliferation was maintained up to seven days in the presence of pullulan. (A) MSC metabolic activity was assessed by quantitative formazan production (A490). MSCs were cultured in 0%, 2% and 5% pullulan for one, three and seven days. An increase in metabolism at day 1 was observed in MSCs cultured in 5% pullulan as compared to 0% controls. Metabolic activity was maintained at days 3 and 7 in all cultures, but consistently increased over time with no significant change due to pullulan treatment. (B) An increase in MSC DNA concentration as assayed by PicoGreen incorporation was observed in all culture conditions between days 1 and 3, with a significant decrease in DNA concentration of MSCs cultured in 2% pullulan after seven days as compared to controls at the same time point. (C) The ratio of metabolic activity normalised to DNA content indicated a non-significant increase in MSC metabolism at day 1 in MSCs exposed to 5% pullulan; however, metabolic activity in all cultures decreased with time, returning to day 1 levels at day 7. Data are presented as the mean ± SD of n = 3 biological replicates generated using triplicate measurements, ** = P ≤ 0.01. MSC mesenchymal stem cell; SD, standard deviation.

Figure 3

MSC surface receptor expression decreased over time in the presence of pullulan. Assessment of CD44, CD90, CD73 and CD105 receptor expression was made by cytometric flow analysis, after one, three and seven days exposure to increasing concentrations of pullulan. (A) There was no significant difference in receptor expression between MSCs incubated in 2% and 5% pullulan as compared to 0% controls after one day. (B) There was a decrease in overall receptor surface expression for all groups after three days of culture. A decrease was observed in expression of CD90, CD73 and CD105 for MSCs cultured in 5% pullulan after three days compared to 0% and 2% treated cultures; however, it was not statistically significant. (C) A further decrease in CD44, CD90, CD105 and a significant decrease in CD73 receptor expression was noted after seven days of MSC culture in 5% pullulan. Data are presented as the mean ± SD of n = 3 biological replicates generated using triplicate measurements, * = P ≤0.05. MSC, mesencgymal stem cells; SD, standard deviation.

Figure 4

MSC differentiation was maintained in the presence of pullulan. Osteogenesis, demonstrated by alizarin red stain showed increased mineral deposition in 2% and 5% pullulan-treated groups as compared to the 0% control. Oil Red O staining of lipid droplets for adipogenesis showed differentiated MSCs in all pullulan-treated groups. Histological assessment by Safranin-O of MSCs in pellet chondrogenic culture showed GAG deposition in all pullulan-treated groups. GAG, glycosaminoglycans; MSCs, mesenchymal stem cells.

Figure 5

Pullulan enhanced MSC retention on OA cartilage. (A) PKH26 labelled MSCs (red) bound to fibrillations on DAPI stained human explanted OA cartilage (blue). MSC retention was increased in (B) 2% or (C) 5% pullulan coated explants. (D) Stereological quantification of MSC retention indicated an increase in cell number on 2% and 5% pullulan coated explants as compared to untreated controls. Magnification bars = 100 μm. DAPI, 4′,6-diamidino-2-phenylindole; MSCs, mesenchymal stem cells; OA, osteoarthritis.

Figure 6

Dectin-2 expression on MSCs was significantly upregulated over time in the presence of pullulan. Histogram plots for MSCs incubated in the presence of increasing pullulan concentrations at 2, 24 and 48 hours (A). Graphs show curve shifts with increasing Dectin-2 staining of cells, with increasing concentrations of pullulan and length of exposure (A). Bar chart representation of Dectin-2 expression on MSCs over time as a result of pullulan exposure (hours) (B). At all time points of exposure, there was an increase in Dectin-2 expression with increasing concentration of pullulan. After 24 and 48 hours, the increase in Dectin-2 expression was significantly increased in 5% pullulan cultures compared to 0% controls. Data are presented as the mean ± SD of n = 3 biological replicates generated using triplicate measurements, ** = P ≤0.01; *** = P ≤0.001 (B). The mean fluorescence intensity (MFI) of Dectin-2 expression was calculated for MSCs following exposure to increasing concentrations of pullulan over 48 hours. The results of three independent experiments are shown (n = 3 donors) ± SEM (C). MSCs, mesenchymal stem cells; SD, standard deviation; SEM, standard error of the mean.