Means of enhancing bone fracture healing: optimal cell source, isolation methods and acoustic stimulation

Abstract

Background: The human body has an extensive capacity to regenerate bone tissue after trauma. However large defects such as long bone fractures of the lower limbs cannot be restored without intervention and often lead to nonunion. Therefore, the aim of the present study was to assess the pool and biological functions of human mesenchymal stromal cells (hMSCs) isolated from different bone marrow locations of the lower limbs and to identify novel strategies to prime the cells prior to their use in bone fracture healing. Following, bone marrow from the ilium, proximal femur, distal femur and proximal tibia was aspirated and the hMSCs isolated. Bone marrow type, volume, number of mononuclear cells/hMSCs and their self-renewal, multilineage potential, extracellular matrix (ECM) production and surface marker profiling were analyzed. Additionally, the cells were primed to accelerate bone fracture healing either by using acoustic stimulation or varying the initial hMSCs isolation conditions.

Results: We found that the more proximal the bone marrow aspiration location, the larger the bone marrow volume was, the higher the content in mononuclear cells/hMSCs and the higher the self-renewal and osteogenic differentiation potential of the isolated hMSCs were. Acoustic stimulation of bone marrow, as well as the isolation of hMSCs in the absence of fetal bovine serum, increased the osteogenic and ECM production potential of the cells, respectively.

Conclusion: We showed that bone marrow properties change with the aspiration location, potentially explaining the differences in bone fracture healing between the tibia and the femur. Furthermore, we showed two new priming methods capable of enhancing bone fracture healing.

Keywords: Acoustic stimulation; Bone marrow; Cell priming; hMSCs.

Fig. 1

Schematic representation of the experimental design. Aspiration of BM from different locations of the lower limb extremities and selection of the optimal cell source, based on hMSC number and phenotype. In vitro priming of hMSC by use of acoustic stimulation or varying the initial culture conditions with the final aim to enhance in vivo bone fracture healing

Fig. 2

Characterization of BM aspirated (BMA) from different locations. a Correlation between aspirated BM volume and MNCs concentration, for the ilium (circle) and proximal femur (square). b Correlation between the plated BM volumes and the number of isolated hMSCs, heterogeneous isolation condition only. c BM viscosity curves from different aspiration locations, represented as correlation between the shear rate and the viscosity. The values represented the mean ± standard deviation of three BM donors (n = 3). Statistically significant differences were found with ***p < 0.001 and **p < 0.01

Fig. 3

Biological characterization of hMSCs isolated from different BM locations. a Proliferation of hMSCs calculated as PD/day from P1 to P2, donor and location dependent. b Proliferation average for all the donors. c CFU potential of hMSCs, donor and location dependent. d CFU average for all the donors. e ECM production, quantification of nodule size area in mm² after cell condensation, donor and location dependent. f ECM production average. g Osteogenic potential calculated as percentage of ALP positive colonies within the CFUs, donor and location dependent. h Osteogenesis average. i Adipogenic potential, quantification of Oil red O staining relative to 100% Oil red O staining solution, donor and location dependent. j Adipogenesis average. The uniform distribution of data, to test inter-donor variation, was assessed using Chi-squared test and presented as a line above all donors. Values are represented as mean ± standard deviation of at least three independent experiments (n ≥ 3). Statistically significant differences were found with ***p < 0.001, **p < 0.01 and *p < 0.05

Fig. 4

Acoustic stimulating device. a Sketch of the fluid flow within the processing chamber and the formation of a standing wave. b Processing chamber. c Speaker, in white, located on the bottom of the processing chamber. d Standing wave pattern formed in bone marrow at 300Hz

Fig. 5

Biological characterization of isolated hMSCs from acoustic stimulated BM at 300 and 500Hz for 5 and 10 min. The results are presented as the fold change over the non-stimulated BM (baseline). a Proliferation of hMSCs calculated as PD/day from P1 to P2, donor and stimulation dependent. b Proliferation average. c CFU potential of hMSCs, donor and stimulation dependent. d CFU average. e ECM production, quantification of nodule size area in mm², donor and stimulation dependent. f ECM production average. g Osteogenic potential calculated as percentage of ALP positive colonies within the CFUs, donor and stimulation dependent. h Osteogenesis average. i Adipogenic potential, quantification of Oil red O staining relative to 100% Oil red O staining solution, donor and stimulation dependent. j Adipogenesis average. Values are represented as mean ± standard deviation of at least three independent experiments (n ≥ 3). Statistically significant differences were found with ***p < 0.001, **p < 0.01 and *p < 0.05

Fig. 6

Biological characterization of hMSCs isolated from BM under different isolation procedures a Proliferation of hMSCs calculated as PD/day from P1 to P2, donor and isolation procedure dependent. b Proliferation average. c ECM production, percentage of formed nodules, donor and isolation procedure dependent. d ECM production average. e Adipogenic potential, quantification of Oil red O staining relative to 100% Oil red O staining solution, donor and isolation procedure dependent. f Adipogenesis average. Values are represented as mean ± standard deviation of at least three independent experiments (n ≥ 3). Statistically significant differences were found with ***p < 0.001 and **p < 0.01