Expanded BMSC here underwent intramembraneous

Expanded BMSC here underwent intramembraneous bone formation on β-TCP, based on an apparent absence of a cartilage intermediate, in line with previous reports (Krebsbach et al., 1997; Martin et al., 1997; Mendes et al., 2002, 2004). Unless subjected to chondrogenic pre-induction, BMSC/β-TCP constructs, however, did not attract haematopoietic marrow, as in our previous study (Janicki et al., 2010), although EZ Cap Reagent GG were expanded in an optimized expansion medium (Dexheimer et al., 2012). This contrasts with findings from other groups (Mankani et al., 2006; Sacchetti et al., 2007) and may be influenced by our expansion medium which contained bFGF and less than 20% fetal calf serum. The altered expansion medium formulation, however, obviously improved ectopic bone formation of pre-induced β-TCP-free BMSC constructs compared to our previous work, in which woven bone was only rarely seen in pre-induced but β-TCP-free BMSC pellets (Janicki et al., 2010; Weiss et al., 2010). Here, we consistently detected bone and hematopoietic marrow formation in pre-induced BMSC constructs in the absence of β-TCP. Further, all pre-induced BMSC/ß-TCP constructs initiated marrow formation in line with previous results (Janicki et al., 2010). bFGF was previously suggested to stimulate cell proliferation and to favor maintenance of stemness in BMSC cultures leading to better bone formation (Martin et al., 1997; Bianchi et al., 2003). In contrast, Sacchetti et al. observed that treatment of BMSC with bFGF did not enhance ectopic bone formation and restricted the differentiation potential of BMSC in a way that haematopoiesis was not supported (Sacchetti et al., 2007). Further studies elucidating the exact influence of bFGF on BMSC, including the effects of dosage and exposure time, with regard to bone formation and capacity to attract haematopoietic marrow are required to better understand the multiple actions of this protein and, further, optimize its use for bone regeneration. Shown repetitively by published studies and supported by our data, ectopic bone formation by transplanted human MSC is a complex development which may fail for several reasons: i) suboptimal maintenance of stemness, ii) insufficient commitment of progenitors to the osteochondral lineage, or iii) an insufficiently osteopermissive microenvironment for bone deposition. Regarding the first point, adequate culture conditions must be fostered for using cells at high proliferation rates (Dexheimer et al., 2012). Regarding the second point, triggering the endochondral pathway of bone formation can efficiently enhance commitment, albeit at the expense of an enhanced culture effort. The third point is well compensated by a calcium phosphate ceramic, providing an enriched microenvironment for bone formation. According to the non-osteogenic tissue origin, human ATSC seem to require all three aforementioned conditions for reproducible ectopic bone formation, while BMSC may well form bone with any of the two combinations. Importantly, chondrogenic pre-induction can be achieved in the absence of animal serum and could, thus, be easily adapted to clinically compliant protocols to profit from easy accessible autologous ATSC in tissue engineering approaches and in regenerative surgery.
Conclusion Our study revealed a lack of ectopic bone forming capacity of human ATSC compared to BMSC, which may be grounded in a reduced osteogenic cell commitment (lower BMP-2 and BMP-4 expression), lower proangiogenic activity (less VEGF, angiopoietin and IL-6) and/or enhanced production of adipose tissue-expressed hormones like visfatin. Multiclonal ATSC populations from liposuction material should, therefore, not be considered skeletal stem cells, since they are not capable of initiating a new bone organ without additional factors inducing a skeletogenic transcriptional program (Bianco et al., 2013). Chondrogenic pre-induction with TGF-ß and BMP-6 on osteo-supportive β-TCP provided an adequate osteopermissive environment to support ectopic bone formation by ATSC. Thus, applying a clinically compliant protocol fulfilling these conditions may render ATSC, with their relatively easy retrieval, high yield and fast proliferation capacity useful for therapeutic approaches to stimulate bone regeneration. However, since these benefits are currently balanced by timely and intensive in vitro culture efforts, use of BMSC may continue to be the more attractive option.