Surgical vacuum filter-derived stromal cells are superior in proliferation to human bone marrow aspirate

Abstract

Background: During joint replacement, surgical vacuum suction guarantees a sufficient overview on the situs. We assume high concentrations of mesenchymal stromal cells (MSCs) on surgical vacuum filters. We compared the in vitro proliferative and differentiation potency of cells from the following: (i) bone marrow (BM), (ii) cancellous bone (CB), (iii) vacuum filter (VF), and (iv) cell saver filtrate reservoir (SF) in 32 patients undergoing elective total hip replacement.

Methods: Mononuclear cells (MNC) were isolated, and cell proliferation and colony-forming units (CFU) were measured. Adherent cells were characterized by flow cytometry for MSC surface markers. Cells were incubated with osteogenic, adipogenic, and chondrogenic stimuli. Cells were cytochemically stained and osteoblastic expression (RUNX-2, ALP, and BMP-2) investigated via qPCR.

Results: Dependent on the source, initial MNC amount as well as CFU number was significantly different whereas generation time did not vary significantly. CFU numbers from VF were superior to those from SR, BM, and CB. The resulting amount of MSC from the respective source was highest in the vacuum filter followed by reservoir, aspirate, and cancellous bone. Cells from all groups could be differentiated into the three mesenchymal lines demonstrating their stemness nature. However, gene expression of osteoblastic markers did not differ significantly between the groups.

Conclusion: We conclude that surgical vacuum filters are able to concentrate tissue with relevant amounts of MSCs. A new potent source of autologous regeneration material with clinical significance is identified. Further clinical studies have to elucidate the regenerative potential of this material in an autologous setting.

Keywords: Bone marrow; Bone regeneration; Cell saver; Colony-forming units; Filter; Mesenchymal stromal cells.

Fig. 1

Tissue harvest during surgery. a Mixture of different liquid tissue components (arrow) released during implantation of a cementless titanium hip stem before aspiration by the surgical vacuum sucker. b Surgical vacuum filter handle: empty and with a vacuum filter clot (VF) (arrow). c Cell saver filtrate reservoir (SR): empty and with collected liquid (arrow). d Femur head before the removal of cancellous bone (CB) (arrow)

Fig. 2

Mononuclear cell (MNC) yield of the different tissue sources. a Total number of harvested MNC of the different tissues. b The number of MNC per gram tissue sample. The figure shows the single values of n = 32 patients as symbols with the median as line. BM bone marrow, CB cancellous bone, VF vacuum filter, SF cell saver filtrate reservoir. Significant differences (Kruskal-Wallis test) are indicated with ****p < 0.0001, ***p < 0.001, and **p < 0.01

Fig. 3

Generation time in days. The figure shows the single values (of n = 27 patients for BM and SR, of n = 28 patients for VF, and of n = 29 patients for CB) as symbols with the median as line. BM bone marrow, CB cancellous bone, VF vacuum filter, SF cell saver filtrate reservoir. There were no significant differences (Kruskal-Wallis test)

Fig. 4

Representative flow cytometry analysis for all groups. Data are shown as a histogram overlay: isotype control (purple) and specific cell surface markers (blue line). Cells were labeled with antibodies against CD34, CD45, CD73, CD90, and CD105

Fig. 5

Characteristic cytochemical staining. Upper row: osteoblast differentiation: staining of calcium in the extracellular matrix components with Alizarin red. The calibration bar indicates 200 μm. Middle row: adipoblast differentiation: staining of intracellular neutral triglycerides with Oil Red. Calibration bar indicates 200 μm. Lower row: chondroblast differentiation: staining of glycosaminoglycans with Alcian blue. The calibration bar indicates 50 μm

Fig. 6

The number of colony-forming units (CFU) per 1 Mill mononuclear cells (MNC) after a cultivation period of 7 days. The figure shows the single values of n = 32 patients as symbols with the median as line. Significant differences (Kruskal-Wallis test) are indicated with ****p < 0.0001

Fig. 7

Mesenchymal stromal cell (MSC) yield of different tissue sources. a Total amount of MSC resulting from the initial mononuclear cell (MNC) number in the different tissue sources. b Amount of MSC per gram tissue weight resulting from the initial MNC number per gram tissue weight. The figure shows the single values of n = 32 patients as symbols with the median as line. BM bone marrow, CB cancellous bone, VF vacuum filter, SF cell saver filtrate reservoir. Significant differences (Kruskal-Wallis test) are indicated with ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05

Fig. 8

Gene expression of the osteoblastic markers. The target genes were normalized to the reference gene GAPDH using the der ΔCt method with ΔCt = Ct test gene − Ct reference gene (GAPDH) and ΔΔCt = ΔCt sample − ΔCt calibrator (unstimulated cells). The relative quantification (RQ) (=fold change compared to the calibrator) was calculated as 2^-ΔΔCt. The figure shows the single values of n = 10 patients as symbols with the median as line. BM bone marrow, CB cancellous bone, VF vacuum filter, SF cell saver filtrate reservoir. a RQ of RUNX. b RQ of ALP. c RQ of BMP-2

Fig. 9

As an outlook for potential clinical application, an innovative surgical sucker handle was designed (EU/EFRE grant 1803su003). It includes a removable filter of beta-tricalcium-phosphate qualified for bone substitution and a biocompatible cylindric handle system with a connector and twist-off cap (BoneFlo®, TissueFlow GmbH, Germany)