The Efficiency of Bone Marrow Aspiration for the Harvest of Connective Tissue Progenitors from the Human Iliac Crest

Abstract

Background: The rational design and optimization of tissue engineering strategies for cell-based therapy requires a baseline understanding of the concentration and prevalence of osteogenic progenitor cell populations in the source tissues. The aim of this study was to (1) define the efficiency of, and variation among individuals in, bone marrow aspiration as a means of osteogenic connective tissue progenitor (CTP-O) harvest compared with harvest from iliac cancellous bone, and (2) determine the location of CTP-Os within native cancellous bone and their distribution between the marrow-space and trabecular-surface tissue compartments.

Methods: Eight 2-mL bone marrow aspiration (BMA) samples and one 7-mm transcortical biopsy sample were obtained from the anterior iliac crest of 33 human subjects. Two cell populations were obtained from the iliac cancellous bone (ICB) sample. The ICB sample was placed into αMEM (alpha-minimal essential medium) with antibiotic-antimycotic and minced into small pieces (1 to 2 mm in diameter) with a sharp osteotome. Cells that could be mechanically disassociated from the ICB sample were defined as marrow-space (IC-MS) cells, and cells that were disassociated only after enzymatic digestion were defined as trabecular-surface (IC-TS) cells. The 3 sources of bone and marrow-derived cells were compared on the basis of cellularity and the concentration and prevalence of CTP-Os through colony-forming unit (CFU) analysis.

Results: Large variation was seen among patients with respect to cell and CTP-O yield from the IC-MS, IC-TS, and BMA samples and in the relative distribution of CTP-Os between the IC-MS and IC-TS fractions. The CTP-O prevalence was highest in the IC-TS fraction, which was 11.4-fold greater than in the IC-MS fraction (p < 0.0001) and 1.7-fold greater than in the BMA fraction. However, the median concentration of CTP-Os in the ICB (combining MS and TS fractions) was only 3.04 ± 1.1-fold greater than that in BMA (4,265 compared with 1,402 CTP/mL; p = 0.00004).

Conclusions: Bone marrow aspiration of a 2-mL volume at a given needle site is an effective means of harvesting CTP-Os, albeit diluted with peripheral blood. However, the median concentration of CTP-Os is 3-fold less than from native iliac cancellous bone. The distribution of CTP-Os between the IC-MS and IC-TS fractions varies widely among patients.

Clinical relevance: Bone marrow aspiration is an effective means of harvesting CTP-Os but is associated with dilution with peripheral blood. Overall, we found that 63.5% of all CTP-Os within iliac cancellous bone resided on the trabecular surface; however, 48% of the patients had more CTP-Os contributed by the IC-MS than the IC-TS fraction.

Fig. 1

Fig. 1-A Anatomical representation of the human iliac crest, with the sites of bone marrow aspiration and autogenous cancellous bone harvest indicated. Fig. 1-B Sagittal view of the iliac crest depicting the transcortical bone core sample (7 mm in diameter) harvested with a customized biopsy needle.

Fig. 2

Fig. 2-A Large field-of-view images (480 image tiles stitched together) of CTPs seeded on 20.5 × 20.5-mm chamber slides and stained with DAPI. The images shown here have been background-corrected (flattening illumination for each tile) and processed for the removal of artifacts. Fig. 2-B Images processed for colony segmentation (red outline) using an automated algorithm. Fig. 2-C Magnified colony (delineated by yellow in Fig. 2-B) indicating quantitative parameters that may be extracted. (Reprinted, with permission, from ASTM F2944-12 Standard Test Method for Automated Colony Forming Unit (CFU) Assays—Image Acquisition and Analysis Method for Enumerating and Characterizing Cells and Colonies in Culture, copyright ASTM International, 100 Barr Harbor Drive, West Conshohocken, PA 19428. A copy of the complete standard may be obtained from ASTM, www.astm.org.)

Fig. 3

The yield of nucleated cells (cellularity) from bone marrow aspiration (BMA) and iliac crest bone, with values for each subject linked by a colored line. IC-TOTAL represents the combined total from the iliac crest marrow space (IC-MS) and trabecular surface (IC-TS) samples. The values are given as millions of nucleated cells per mL of sample.

Fig. 4

The yield of nucleated cells (cellularity) from the marrow space (IC-MS) and trabecular surface (IC-TS) fractions obtained from the iliac crest bone, with values for each subject linked by a colored line. The values are given as millions of nucleated cells per mL of sample.

Fig. 5

The prevalence of CTP-Os from bone marrow aspiration (BMA) and iliac crest bone core, with values for each subject linked by a colored line. IC-TOTAL represents the combined total from the iliac crest marrow space (IC-MS) and trabecular surface (IC-TS) samples. The values are given as the number of CTP-Os per million nucleated cells plated.

Fig. 6

The prevalence of CTP-Os in the marrow space (IC-MS) and trabecular surface (IC-TS) fractions obtained from the iliac crest bone, with values for each subject linked by a colored line. The values are given as the number of CTPs per million nucleated cells plated.

Fig. 7

The concentration of CTP-Os from bone marrow aspiration (BMA) compared with iliac crest bone core, with values for each subject linked by a colored line. IC-TOTAL represents the combined total from the iliac crest marrow space (IC-MS) and trabecular surface (IC-TS) samples. The values are given as the number of CTP-Os per mL of tissue.

Fig. 8

The concentration of CTP-Os in the marrow space (IC-MS) and trabecular surface (IC-TS) fractions obtained from the iliac crest bone, with values for each subject linked by a colored line. The values are given as the number of CTP-Os per mL of tissue.