Hypoxia Impedes Hypertrophic Chondrogenesis of Human Multipotent Stromal Cells

Abstract

Within the field of bone tissue engineering, the endochondral approach to forming bone substitutes represents a novel concept, where cartilage will undergo hypertrophic differentiation before its conversion into bone. For this purpose, clinically relevant multipotent stromal cells (MSCs), MSCs, can be differentiated into the chondrogenic lineage before stimulating hypertrophy. Controversy exists in literature on the oxygen tensions naturally present during this transition in, for example, the growth plate. Therefore, the present study focused on the effects of different oxygen tensions on the progression of the hypertrophic differentiation of MSCs. Bone marrow-derived MSCs of four human donors were expanded, and differentiation was induced in aggregate cultures. Normoxic (20% oxygen) and hypoxic (5%) conditions were imposed on the cultures in chondrogenic or hypertrophic differentiation media. After 4 weeks, the cultures were histologically examined and by real-time polymerase chain reaction. Morphological assessment showed the chondrogenic differentiation of cultures from all donors under normoxic chondrogenic conditions. In addition, hypertrophic differentiation was observed in cultures derived from all but one donor. The deposition of collagen type X was evidenced in both chondrogenically and hypertrophically stimulated cultures. However, mineralization was exclusively observed in hypertrophically stimulated, normoxic cultures. Overall, the progression of hypertrophy was delayed in hypoxic compared with normoxic groups. The observed delay was supported by the gene expression patterns, especially showing the up-regulation of the late hypertrophic markers osteopontin and osteocalcin under normoxic hypertrophic conditions. Concluding, normoxic conditions are more beneficial for hypertrophic differentiation of MSCs than are hypoxic conditions, as long as the MSCs possess hypertrophic potential. This finding has implications for cartilage tissue engineering as well as for endochondral bone tissue engineering, as these approaches deal with, respectively, the inhibition or enhancement of hypertrophic chondrogenesis.

Engineering cartilage or endochondral bone: A comparison of different naturally derived hydrogels

“…This was would suggest that relative levels of type X collagen production preimplantation are not necessarily predictive of the extent of endochondral bone formation in vivo. The more rapid degradation rate of the alginate in vivo is likely favoring vascularization and oxygen availability within this hydrogel, and subsequently driving hypertrophy [30,46].…”

Endochondral bone formation in gelatin methacrylamide hydrogel with embedded cartilage-derived matrix particles

“…The multilineage potential of the cells that were cultured from the bone marrow aspirate was investigated by a three-way differentiation assay as previously described [19,28,29].…”

A computational model to explore the role of angiogenic impairment on endochondral ossification during fracture healing

“…This computational model did not however propose a specific hypothesis for how environmental factors regulate the initiation and progression of chondrocyte hypertrophy and endochondral ossification, which is central to successful secondary fracture healing. This is clearly a potential limitation of such models, as a number of recent studies have demonstrated that low oxygen conditions can supress hypertrophy and endochondral ossification of chondrogenically primed MSCs (Zhu et al, 2014;Sheehy et al, 2012;Gawlitta et al, 2012;Leijten et al, 2012Leijten et al, , 2014.…”

“…Motivated by in-vitro observations (Zhu et al, 2014;Sheehy et al, 2012;Gawlitta et al, 2012;Leijten et al, 2012Leijten et al, , 2014 our previous in-silico model of MSC differentiation (Burke and Kelly, 2012) was updated to include specific rules for how oxygen levels regulate hypertrophy of chondrocytes. The model was then tested by attempting to simulate fracture healing within a murine tibia in both normal and ischemic limbs, where experimental data was generated to quantify the proportion of hypertrophic cartilage within the calluses of such bones at specific time-points after the initiation of injury.…”