Human CD34+ stem cells produce bone nodules in vivo

Abstract

Objectives: The aim of this study was to select and provide enough stem cells for quick transplantation in bone engineering procedures, avoiding any in vitro expansion step.

Materials and methods: Dental germ pulp, collected from 25 healthy subjects aged 13-20 years, were subjected to magnetic-activated cell sorting to select a CD34(+) stem cell population capable of differentiating into pre-osteoblasts. These cells were allowed to adhere to an absorbable polylactic-coglycolic acid scaffold for 30 min, without any prior expansion, and the CD34(+) cell-colonized scaffolds were then transplanted into immunocompromised rats, subcutaneously.

Results: After 60 days, analysis of recovered transplants revealed that they were formed of nodules of bone, of the same dimensions as the original scaffold. Bone-specific proteins were detected by immunofluorescence, within the nodules, and X-ray diffraction patterns revealed characteristic features of bone. In addition, presence of platelet endothelial cell adhesion molecule and von Willebrand factor immunoreactivity were suggestive of neo-angiogenesis and neovasculogenesis taking place within nodules. Importantly, these vessels were HLA-1(+) and, thus, clearly human in origin.

Conclusions: This study indicates that CD34(+) cells obtained from dental pulp can be used for engineering bone, without the need for prior culture expanding procedures. Using autologous stem cells, this schedule could be used to provide the basis for bone regenerative surgery, with limited sacrifice of tissue, low morbidity at the collection site, and significant reduction in time needed for clinical recovery.

Figure 1

Flow cytometric analysis of cells, performed after digestion before (a) and after (b) CD34 MACS selection. CD34⁺ cells were also positive for CD117 (c), CD90 and CD133 (d).

Figure 2

Immunofluorescence confirming the presence of HLA‐1 (a) (original magnification ×200) and of a mineralized extracellular matrix of the transplant. The panel shows positivity for BSP (b) (original magnification ×200), OC (c) (original magnification ×100), BAP (d) (original magnification ×200) and ON (e) (original magnification ×200).

Figure 3

Immunofluorescence showing platelet/endothelial cell adhesion molecule‐1 (PECAM‐1), von Willebrand factors 1/2 and HLA‐1 positive vessels within bone obtained after 60 days of transplantation. (a) PECAM‐1 positivity and (b) DAPI counterstaining (Original magnification ×200); (c) von Willebrand factor 1/2 positivity and (d) DAPI counterstaining (Original magnification ×100); (e) HLA‐1 positivity and (f) DAPI counterstaining (Original magnification ×400); (g) to identify the presence of human cells within mouse tissues, α‐mod17 PCR has been performed. Lane 1: 3T3 cells (mouse fibroblasts); lane 2: human gingival fibroblasts; lane 3–6: 1–4 test samples. (h) FACS analysis showing HLA‐1 positivity in cells obtained from transplants at both sacrifice times.

Figure 4

Haematoxylin and eosin staining. In vivo sample produced with CD34⁻ cells. No bone nodules were formed, only connective tissue (red arrow) and PLGA (black arrow) can be observed (original magnification ×200).

Figure 5

Haematoxylin and eosin staining. In vivo sample produced with CD34⁺ cells. (a) Sample after 30 days, in which small fragments of non‐absorbed PLGA (black arrow) and a fibrous bone tissue (red arrow) are present (original magnification ×400). (b) Sample after 60 days showing remodelled, newly formed bone (arrow) (original magnification ×200).

Figure 6

X‐ray diffraction analysis of a sample of mandibular cortex (in black) and of a 60‐day‐old transplant (in green). Typical crystals of human bone can be seen. The diffraction X‐ray diffraction pattern is completely superimposable onto that of the control sample (in black). Different positions on the Y axis of the two samples are due to immaturity of the tissue. Analyses were repeated in quadruplicate without appreciable differences between samples.