Impact of Age on Human Adipose Stem Cells for Bone Tissue Engineering

Abstract

Bone nonunion is a pathological condition in which all bone healing processes have stopped, resulting in abnormal mobility between 2 bone segments. The incidence of bone-related injuries will increase in an aging population, leading to such injuries reaching epidemic proportions. Tissue engineering and cell therapy using mesenchymal stem cells (MSCs) have raised the possibility of implanting living tissue for bone reconstruction. Bone marrow was first proposed as the source of stem cells for bone regeneration. However, as the quantity of MSCs in the bone marrow decreases, the capacity of osteogenic differentiation of bone marrow stem cells is also impaired by the donor's age in terms of reduced MSC replicative capacity; an increased number of apoptotic cells; formation of colonies positive for alkaline phosphatase; and decreases in the availability, growth potential, and temporal mobilization of MSCs for bone formation in case of fracture. Adipose-derived stem cells (ASCs) demonstrate several advantages over those from bone marrow, including a less invasive harvesting procedure, a higher number of stem cell progenitors from an equivalent amount of tissue harvested, increased proliferation and differentiation capacities, and better angiogenic and osteogenic properties in vivo. Subcutaneous native adipose tissue was not affected by the donor's age in terms of cellular senescence and yield of ASC isolation. In addition, a constant mRNA level of osteocalcin and alkaline phosphatase with a similar level of matrix mineralization of ASCs remained unaffected by donor age after osteogenic differentiation. The secretome of ASCs was also unaffected by age when aiming to promote angiogenesis by vascular endothelial growth factor (VEGF) release in hypoxic conditions. Therefore, the use of adipose cells for bone tissue engineering is not limited by the donor's age from the isolation of stem cells up to the manufacturing of a complex osteogenic graft.

Keywords: adipose stem cells; aging; bone; functionality; mesenchymal stem cells.

Fig. 1.

(A, B) Impact of age on adipose stem cells differentiation to obtain a 3-dimensional (3D) scaffold-free and oteogenic graft. (A) No impact of the donor’s age was found on the time required to isolate and expand adipose stem cells (from native adipose tissue) and to obtain finally differentiated to the scaffold-free 3D osteogenic graft. The integrity of the 3D graft was histomorphologically assessed at the end of manufacturing by the potency score (before implantation). A score of the graft integrity (for the optimal 3D graft) between −1 and +1 in terms of cell viability counting, interconnective tissue integrity, and demineralized bone matrix content. Note that a final 3D graft was always obtained (individual donors without any impact of donor age, which ranged from 5 and 70 y) for adipose-derived stem cells incubated with demineralized bone matrix, demonstrating the reproducibility of the manufacturing procedures.

Fig. 2.

Impact of donor’s age on the integrity of the scaffold-free 3-dimensional (3D) graft. Macroscopically, the 3D osteogenic graft was obtained for donors aged below 18 y old and older 60 y old (upper level). Microscopically, the collagen matrix, the cellularity with 4′,6-diamidino-2-phenylindole (DAPI) staining (middle level, original magnification: ×10), and the osteocalcin expression (lower level, original magnification: ×25) were maintained in the 3D graft for young and old donors of adipose tissue for adipose-derived stem cell isolation, proliferation, and differentiation.