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In regenerative medicine, there is an approach to avoid expansion of the mesenchymal stem cell (MSC) before implantation. The aim of this study was to compare methods for instant MSC therapy by use of a portable, automatic and closed system centrifuge that allows for the concentration of MSCs. The main outcome measures were the amount of MSCs per millilitre of bone marrow (BM), clusters of differentiation (CD), proliferation and differentiation capacities of the MSC. A volume reduction protocol was compared to the traditional laboratory methods of isolation using a Ficoll gradient and native BM. Fifty millilitres of BM were obtained from haematologically healthy male Caucasians (n= 10, age 8 to 49 years). The number of colony forming units-fibroblast (CFU-F)/ml BM was highest in the centrifuge volume reduction protocol, followed by the native BM (not significant), the centrifuge Ficoll (p= 0.042) and the manual Ficoll procedure (p=0.001). The MSC of all groups could differentiate into the mesenchymal lineages without significant differences between the groups. The CD pattern was identical for all groups: CD13+; CD 44+; CD73 +; CD90+; CD105+; HLA-A,B,C+; CD14-; CD34-; CD45-; CD271-; HLA-DR-. In a further clinical pilot study (n=5) with 297 ml BM (SD 18.6), the volume reduction protocol concentrated the MSC by a factor of 14: there were 1.08 x 10 2 MSC/ml BM (standard deviation (SD) 1.02 x 10 2) before concentration, 14.8 x 10 2 MSC/ ml BM (SD 12.4 x 10 2) after concentration, and on average 296 x 10 2 MSC (SD 248.9 x 10 2 , range 86.4-691.5 x 10 2) were available for MSC therapy. The volume reduction protocol of the closed centrifuge allows for the highest concentration of the MSC, and therefore, is a promising candidate for instant stem cell therapy.

The in vitro effect of platelet-rich plasma (PRP) on cell loading, proliferation, and osteogenic differentiation of human mesenchymal stem cells (MSC) is assessed on distinct resorbable and synthetic calcium phosphate scaffolds. A high specific surface area scaffold composed of calcium-deficient hydroxyapatite (CDHA; 48m2/g) is compared with one made out of beta-tricalcium phosphate (beta-TCP; surface area <0.5 m2/g). Fivefold concentrated fresh PRP is applied to scaffolds loaded with 2 x 10(5) MSC (n = 5). These constructs are kept in a medium with osteogenic supplements for 3 weeks. The addition of PRP leads to a higher cell loading efficiency of MSC on CDHA (p = 0.0001), that reaches the values of beta-TCP. Proliferation over 21 days is improved by PRP both on CDHA (p = 0.0001) and beta-TCP (p = 0.014) compared to MSC/calcium phosphate composites. Without the addition of PRP, CDHA has a lower cell loading efficiency (p= 0.0001) and proliferation (p= 0.001) than beta-TCP. The ALP activity is higher in the MSC/ceramics groups than in the monolayer controls (p<0.05). The addition of PRP does not significantly affect ALP activity. However, ALP activity varies considerably within the cell donors and different PRP-pools (p = 0.001), while the cell numbers do not vary within these two parameters. PRP generates a positive effect on the loading efficiency of MSC on the high specific surface scaffold CDHA that thereby reaches the loading efficiency of beta-TCP. PRP improved proliferation, but its osteogenic properties on both calcium phosphate scaffolds are weak.

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