Furthermore a dramatic shift in MSC marker expression was se

Furthermore, a dramatic shift in MSC marker expression was seen, including an increased positivity for CD34 (Wood et al., 1997; Fina et al., 1990; Krause et al., 1994). There is evidence that CD34 is a marker for diverse progenitors including MSCs (Copland et al., 2008). In the current study, mRNA transcript levels of CD34 were higher in freshly isolated hPDCs grown to confluency. This indicates that native progenitors from the periosteum may be CD34+, but the expression is lost upon in vitro expansion. This is in line with previous reports with murine bone marrow mononuclear progenitors, defined as a potent osteoprogenitor cell population, losing their CD34+ upon in vitro expansion (Abdallah et al., 2015). Moreover, the CD34+ cell population displayed an enhanced expression and secretion of osteoprogenitor-secreted factors (Majka et al., 2001; Janowska-Wieczorek et al., 2002). In addition, an elevated expression of BMP type 1 and 2 receptors was seen, suggesting the generation of a more BMP-responsive cell population (Hidalgo et al., 2012). In line with these findings, CDM pre-conditioned hPDCs displayed an earlier onset of, as well as an elevated osteochondrogenic response to BMP treatment, confirmed by BMP-2, -4, -6, -7, -9, and GDF5 stimulation. This improved differentiation potential could be correlated to the CD34+ cell population. In addition, an altered activation of downstream signaling pathways was confirmed in the CDM pre-conditioned cells, suggesting a potential mechanism to the more potent differentiation. Importantly, a change in cellular phenotype and improved differentiation potential was observed in GDC-0994 isolated from donors of different gender and age, in line with previous reports on the osteochondrogenic capacity of PDCs (Chang et al., 2014). Of note, a reduced response was seen in cells isolated from a 47-year-old female, which was associated with low expression levels of BMP receptors. Further screening of more samples will be required to determine whether this is a combined age- and gender-dependent phenomenon (Nicks et al., 2010). Since both an elevated osteogenic as well as chondrogenic differentiation was seen, IHC for SOX9 and OSX was performed to gain information at the single-cell level. Remarkably, CDM pre-conditioned cells displayed a positivity for both markers. Cell-fate studies have revealed that SOX9-expressing limb bud mesenchymal cells give rise to both chondrocytes and osteoblasts, where SOX9 expression precedes RUNX2 and OSX expression during limb bud development (Akiyama et al., 2005; Nakashima et al., 2002). As a consequence, the combined SOX9 and OSX positivity may reflect the stage when cells enter the hypertrophic or early osteoblast stage and undergo a transition from SOX9+ to OSX+ cells. On the other hand, additional markers such as ACAN, COLL10A1, RUNX2, and OCN are also upregulated, further opening the possibility of cells undergoing a dual- or trans-differentiating pathway (Yang et al., 2014; Zhou et al., 2014). Nevertheless, the elevated differentiation capacity seen in vitro could be correlated to an elevated cartilaginous tissue production in vivo. To further investigate the in vivo potential, a biomimetic system in the form of cell aggregation was introduced (Moreira Teixeira et al., 2012; Evans et al., 2013). Initially, an optimal size of the aggregate was determined and an increased aggregate size led to elevated expression of RUNX2, ALP, OSX, and VEGF. This could potentially be explained by the elevated forces exerted by the increased number of cells in the aggregates, which further steered differentiation toward the endochondral fate (Qi et al., 2008; Mammoto et al., 2011). Upon ectopic assessment in vivo, a synergistic effect of aggregation and BMP-2 treatment displayed an enhanced microvessel ingrowth along with the presence of GAG-rich matrix and active BMP signaling. It can be speculated that this was due to endogenous BMP production suggested by an elevated BMP-2 protein secretion, further supported by elevated BMP-2 expression on an mRNA transcript level. Therefore, the endogenous BMP secretion may further have contributed to in vivo endochondral bone formation (Einhorn, 1998; Schindeler et al., 2008). After 3 weeks in vivo, the presence of hypertrophic chondrocytes, cartilage remodeling, and degradation was observed in constructs where simultaneous stimulation by aggregation and BMP-2 was applied. At week 6, these constructs had further developed into bone ossicles. Assessment in a critical size long-bone defect displayed successful bone bridging 4 weeks post transplantation. Qualitative analysis at 2 weeks displayed a process resembling endochondral bone healing with the contribution of the transplanted hPDCs to both the intermediate cartilage tissue as well as the bridging bone. A reduced contribution of the implanted cells was seen at week 8, suggesting that host-derived cells are involved in the remodeling of the newly formed bone tissue.