The importance of physical factors in the development and maintenance of bone tissue has been recognized for many years (Harada and Rodan 2003
) and it has been convincingly shown that bone cells are sensitive to physical stimuli. Recently, tensile forces of up to 4,300 μstrain were reported to support osteogenic differentiation of hMSCs under appropriate culture conditions (Mirza et al. 2007
), which is in accordance with our findings, where cyclic tensile strain of up to 3,000 μstrain was sufficient to initiate an osteogenic stimulus at the gene expression level and at the level of ALP activity ( and ). With regard to biological regeneration, cell differentiation and the number of cells are the critical factors for an appropriate response, and—since CTS affected both factors—the phenotypic osteogenic response to CTS may be best reflected by ALP activity at the culture-well level (μM/min). Interestingly, the hMSCs did not respond well to mechanical strain in some of the patients, while this osteogenic response was quite pronounced in others. Most importantly, this variability was found to be highly related to the BMI of the donor ().
The pronounced intrinsic heterogeneity of multipotent hMSCs is well known and is thought to represent the lineage hierarchy, where some of the cells are multipotent stem cells while others are more restricted primitive progenitor cells of several cell lineages at various stages of differentiation (Aubin and Triffitt 2002
). Thus, it might be speculated that the BMI-related mechano-response we found may be due to a commitment of hMSCs independently of CTS, for example, simply due to the number of fat cells in the culture of obese patients. However, this can be ruled out for several reasons: it is very unlikely that cells at passage 4–6 cells would be committed in a cell-specific differentiation pathway. This has been convincingly demonstrated by large-scale gene expression analyses (Tremain et al. 2001
, Kulterer et al. 2007
, Scheideler et al. 2008
) and is further supported by the fact that we detected no messages for adipo- or chondrocytic markers (i.e. CSPCP and COL2A1) in CON and CTS. Most importantly, due to the study design, any commitment independent of CTS would also have affected the hMSCs in CON; but none of the genotypic and phenotypic parameters in CTS and CON (raw data) were related to the mechano-response as determined by paired analysis, or were significantly correlated to any of the donor's physiological characteristics (). Consequently, our findings suggest that physiological factors associated with donor's BMI may not necessarily affect the overall osteogenic potential of hMSCs, but will severely hamper the osteogenic response of hMSCs to mechanical strain, which may be of clinical importance.
It is well established that mechanical forces are major determinants of bone mass (Harada and Rodan 2003
). Although absolute bone density is known to be higher in obese individuals, it has been demonstrated that when adjusted for body weight, these individuals suffer lower relative bone area and bone mass than non-obese individuals (Goulding et al. 2000
), which may also be responsible for a paradoxical increase in fracture risk (Strotmeyer et al. 2005
). This is in accordance with previous findings (Weiler et al. 2000
), which showed that body fat (relative to weight) negatively influenced bone mineral content and bone density during growth, thus compromising the attainment of peak bone mass. In addition, there is evidence of increased risk of implant failure after lower limb joint surgery in obese patients, particularly after total joint replacement of the knee (Winiarsky et al. 1998
, Foran et al. 2004
, Berend et al. 2005
, Amin et al. 2006
It is important to consider that all hMSC samples were isolated from bone marrow at the iliac crest. Thus, it seems unlikely that local conditions related to primary diagnosis indicating the need for elective surgery would account directly for the mechano-resistance in our cohort. Indeed, the process of aging as well as obesity itself are accompanied by multiple changes in an individual's whole body physiology, whereas each of them has the potential to severely affect the functional properties of hMSCs within the marrow “stem cell niche” (Caplan et al. 1998
). Although interpretation of the data may be limited due to the small sample size, some of the results found from correlation analysis are quite coherent and provide some tentative suggestions regarding this challenging issue. Indeed, adipose tissue is being increasingly recognized as a secretory organ that releases bioactive peptides known to affect a number of physiological functions in bone metabolism also. As expected, fasting leptin levels correlated with BMI, but were also inversely related to the phenotypic ALP response (), which contrasts with previous findings that leptin is capable of directly stimulating osteoblast differentiation via leptin receptors (Thomas and Martin 2005
). Hence, the inverse correlation between the CTS response and leptin levels is likely to reflect its co-linearity with BMI. Based on our findings, the adipokines adiponectin and resistin cannot be attributed to the BMI-linked mechano-response too, but a weak direct correlation was found with the levels of 17β-estradiol. This is an interesting finding because estrogen is known to be a strong determinant of bone mass in both women and men, and has been convincingly shown in vitro and in vivo to critically mediate mechanostimulatory effects via estrogen receptor (ER)-alpha (Lee et al. 2003
). However, the correlation with the osteogenic mechano-response was weak; thus, as yet unknown variables related to the donor's fat metabolism are likely to play crucial roles in the impaired responsiveness of hMSCs to mechanical stimuli.
While our study was designed with a view to exclusion of covariate factors beyond mechanical straining, we should mention that weak associations may be easily missed due to the sample size and the intrinsic variability of undifferentiated hMSCs. Although causality cannot be proved by correlation analysis, the finding that the variability in genotypic and phenotypic characteristics of hMSCs was not related to BMI—while this was a strong predictor of the phenotypic osteogenic mechano-response under controlled in vitro conditions—is strongly suggestive of a systemically upstream process of imprinting of hMSCs within the donor's marrow stem cell niche. For this, further investigation is necessary, since obesity-related mechano-resistance of hMSCs may possibly be of clinical significance for disturbances in bone metabolism, bone-healing, and the risk of implant failure.