Shed blood-derived cells from total hip arthroplasty have osteoinductive potential: a pilot study

Abstract

Background: Cell therapy using autologous cells has been used in the treatment of various medical conditions. The mononuclear cell (MNC) fraction of bone marrow (BM) contains stem/progenitor cells that could contribute to osteogenesis and angiogenesis.

Questions/purposes: We asked whether MNCs derived from intraoperative shed blood (SB), consisting of peripheral blood and BM, have osteoinductive and angiogenic potential.

Methods: We harvested SB and BM from six patients undergoing THA. Isolated MNCs from SB and BM were analyzed by flow cytometry to evaluate the CD34(+) cell fraction and 1 × 10(6) cells were seeded on an interconnective porous calcium hydroxyapatite ceramic (IP-CHA) and transplanted in the backs of athymic rats. IP-CHAs without cells were transplanted as controls and all composites were harvested after 4 and 8 weeks. Osteoinductive potential was evaluated by histologic observation, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) using anti-osteocalcin (OC) antibodies qualitatively and quantitatively. To evaluate angiogenic potential, capillary density was measured by immunohistochemistry using Isolectin B4 4 weeks after implantation.

Results: We found that CD34(+) cells existed in SB-MNCs and there was a trend toward lower frequency compared with BM-MNCs. Histologic osteoinduction, OC expression, and capillary density were increased by transplantation of MNCs from SB. Similar results were achieved with MNCs from BM.

Conclusions: MNCs from SB have equivalent osteoinductive and angiogenic potential compared with those from BM.

Clinical relevance: SB could be an attractive source for isolation of MNCs, enhancing osteoinduction and neovascularization, to augment the reconstruction of skeletal defects.

Fig. 1

A flow diagram of the experiments (cell preparation and in vivo transplantation) is shown. Bone marrow (BM) and shed blood (SB) are harvested from patients during THA. Isolated mononuclear cells (MNCs) (1 × 10⁶) from BM and SB are seeded on interconnective porous calcium hydroxyapatite ceramic (IP-CHA) and transplanted in the backs of athymic rats for one biochemical and two histologic analyses. IP-CHAs without cells were transplanted as controls. There were three groups and six subjects per group in this study.

Fig. 2A–B

(A) Composites (disk-shaped with a diameter of 5 mm and a height of 2 mm) were sectioned (5 μm) horizontally using a microtome and the center portion of the composite was stained. (B) The newly formed bone and capillary were assessed in five areas (one center and four peripheral per section) of 1.0 mm².

Fig. 3A–B

(A) The frequencies of CD34⁺/CD45^dim cells were evaluated in the bone marrow (BM) and shed blood (SB) groups. A dot plot shows the representative flow cytometric analysis. (B) The percentage of CD34⁺/CD45^dim cells in SB showed lower tendency compared with BM. However, there was no difference between the BM and SB groups (BM group, 2.86% ± 1.03%; SB group, 2.42% ± 0.82%; p = 0.6991 for BM versus SB).

Fig. 4A–D

Histologic observation revealed fibrous tissues and bone formation in composites harvested at 8 weeks after implantation in each group. In the (A) control group, only fibrous tissues were seen although substantial bone formations in the pores of the composite were identified in the (B) bone marrow (BM) and (C) shed blood (SB) groups. (H = interconnective porous calcium hydroxyapatite ceramic (IP-CHA); F = fibrous tissue; L = lamellar bone) (A–C: Stain, hematoxylin and eosin; original magnification, ×400; scale bar = 100 μm). (D) A graph shows the percentage of newly formed bone in the composite 8 weeks after implantation. Compared with the control group, the percentage was higher in the BM and SB groups (control group, 10.4% ± 6.7%; BM group, 26.8% ± 4.9%; SB group, 25.9% ± 3.1%; p = 0.0150 for control versus BM; p = 0.0065 for control versus SB; p = 1.0000 for BM versus SB).

Fig. 5A–D

Lamellar bone and lining osteocalcin (OC)-positive osteocytes are identified in the bone marrow (BM) and shed blood (SB) groups 8 weeks after implantation in the (A) control, (B) BM, and (C) SB groups. [H = interconnective porous calcium hydroxyapatite ceramic (IP-CHA); F = fibrous tissue; L = lamellar bone, white arrows: lining OC-positive osteocytes](Stain, immunochemical stain; original magnification, ×400; scale bar = 100 μm). (D) Four and 8 weeks after implantation, the expression level of OC was higher in the BM and SB groups compared with the control group. There was no difference between the BM and SB groups at either time (4 weeks: control group, 18.6 ± 3.5 ng/mL; BM group, 36.8 ± 3.2 ng/mL; SB group, 39.5 ± 9.0 ng/mL; p = 0.0065 for control versus BM; p = 0.0260 for control versus SB; p = 0.3961 for BM versus SB) (8 weeks: control group, 22.2 ± 10.9 ng/mL; BM group, 50.0 ± 6.5 ng/mL; SB group, 49.4 ± 19.2 ng/mL; p = 0.0065 for control versus BM; p = 0.0455 for control versus SB; p = 1.0000 for BM versus SB).

Fig. 6A–D

Capillaries were identified as blood vessel endothelium positive for Isolectin B4 in the (A) control, (B) bone marrow (BM), and (C) shed blood (SB) groups (Stain, immunohistochemical stain; original magnification, ×400; scale bar = 100 µm). (D) The density of capillaries was greater in the BM and SB groups compared with the control group at 4 weeks. There was no difference between the BM and SB groups (control group, 104 ± 7.1/mm²; BM group, 215 ± 19.4/mm²; SB group, 217 ± 25.5/mm²; p = 0.0150 for control versus BM; p = 0.0150 for control versus SB; p = 1.0000 for BM versus SB).