Multipotential Stromal Cell Abundance in Cellular Bone Allograft: Comparison with Fresh Age-Matched Iliac Crest Bone and Bone Marrow Aspirate

Figures & data

Figure 1. Phenotypic and functional characterization of cells grown from Osteocel^® explant cultures.

(A) Proliferative and (B) differentiation capacities, chondrogenesis and adipogenesis were assessed on day 21 post-induction by Toluidine Blue or Oil Red-O staining, respectively. Osteogenesis was assessed by alkaline phosphatase staining on day 14 post-induction; original magnification of microphotographs ×100 and ×40 for chondrogenesis. (C) Phenotypic characterization of Osteocel-derived cells confirming their identity as MSCs (n = 3 Osteocel lots). Gray filled histograms indicate isotype control staining. (D–F) Immunosuppressive capacity of Osteocel-MSCs in co-culture with T cell responders (Tresp) CD4⁺ T cells. (D) Gating strategy for Tresp cell employing initial gating for live cells followed by exclusion of MSCs and final gating on the CD4⁺ population. (E) Histograms indicating proliferation patterns of Tresp co-cultured at ratios 1:1 to 1:4 with MSCs (a representative donor, colors represent cell doublings). (F) Inhibition of Tresp proliferation measured as relative division index normalized to no MSC control (n = 4 Osteocel lots, 1:1 to 1:8 ratios). Error bars represent SD.

FI: Fluorescence intensity; MSC: Multipotential stromal cell; SSC: Side scatter.

Figure 2. Assessment of cell abundance in Osteocel^® as analyzed by histology, confocal microscopy and light microscopy.

(A) Hematoxylin and eosin staining of decalcified fragments of Osteocel bone; compared with (B) control iliac crest bone; black filled arrows indicate presence of bone-lining cells, white filled arrows indicate embedded octeocytes. (C) Confocal microscopy indicating the presence of live (green) and dead (red) cells on the surface of Osteocel and (D) following the removal of bone reflected light signal showing more live cells embedded in the bone matrix (osteocytes), similar to (A). (E) Abundant osteocytes stained with Crystal Violet, original magnification ×40 and (F) ×100 indicated by white filled arrows.

Figure 3. Environmental scanning electron microscopy of stromal cell abundance (Day 0, top panels) and in vitro scaffold colonization (Day 21, middle and bottom panels) in Osteocel^® and control iliac crest bone.

White filled arrows show cell attachment points to bone, fat cells are indicated by dotted outline and black filled arrows.

Figure 4. Analysis of CD45^-CD271⁺ multipotential stromal cell abundance in femoral head bone digests.

(A) New live/dead binding dyes compared with (B) a traditional 7-AAD-based method. Far left panels: dot plots showing size distribution of all events (cells and debris). Middle left panels: gating on live cells (shown in red). Middle right panels: identification of CD45^-CD271⁺ MSCs (shown in blue). Far right panels: histogram of CD73 positivity on the CD45^-CD271⁺ MSC population. Vertical lines indicate the boundary for isotype control binding, horizontal lines indicate the region used to calculate the proportions of CD73⁺ cells.

FSC: Forward scatter; MSC: Multipotential stromal cell; SSC: Side scatter.

Figure 5. Analysis of CD45^-CD271⁺ multipotential stromal cell abundance in enzymatic digests of Osteocel^® and control age-matched iliac crest bone.

(A) Analysis of CD45^-CD271⁺ MSC abundance in enzymatic digests of Osteocel^® and (B) control age-matched IC bone. (A & B) Left panels show the abundance of CD45^-CD271⁺ MSCs and hematopoietic lineage cells, in (A) Osteocel and (B) IC bone, respectively. (A & B) Middle panels show CD73 positivity of CD45^-CD271⁺ MSCs, shown as histograms. Vertical lines indicate the boundary for isotype control binding, horizontal lines indicate a region used to calculate the proportions of CD73⁺ cells. (C) CD90 (n = 8 lots), CD105 (n = 5 lots) and CD73 (n = 11 lots) expression on CD45^-CD271⁺ cells from Osteocel (light blue bars) compared with IC bone control (dark blue bars; n = 4, four and five donors, respectively). (D) ImageStream™ morphological analysis of CD45^-CD271⁺ in a representative Osteocel digests collected in the ‘live cell‘ gate (top) and debris/demineralized bone matrix gate (bottom). (E) Absolute numbers of CD45^-CD271⁺ MSCs in Osteocel, control IC bone and bone marrow aspirate per 1 g (or 1 ml) indicating inferiority of bone marrow aspirate compared with both IC bone and Osteocel (both significant at p < 0.0001). Error bars represent standard deviation.

HLC: Hemopoietic lineage cell; IC: Iliac crest; MSC: Multipotential stromal cell.

Table 1. Molecular analysis of Osteocel^® in comparison to dermal fibroblast and osteoblast cell lines.

Full name	Gene code	Versus osteoblasts^†	Versus fibroblasts^†
Osteopontin, transcript variant 2	SPP1	22.3	119.7
Osteopontin, transcript variant 1	SPP1	26.0	64.2
Chemokine (C-X-C motif) receptor 7, transcript variant 1	CXCL7	29.4	63.3
S100 calcium binding protein A8	S100A8	88.3	62.8
Transferrin	TF	30.8	54.3
Insulin-like growth factor binding protein 3, transcript variant 1	IGFBP3	1.5	38.1
Bone γ-carboxyglutamate (gla) protein (osteocalcin)	BGLAP	26.6	36.8
Vascular cell adhesion molecule 1, transcript variant 1	VCAM1	7.8	25.9
S100 calcium binding protein A12	S100A12	16.3	22.4
Chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1), transcript variant 1	CXCL12	-3.3	21.2
Frizzled-related protein	FRZB	15.4	17.00
S100 calcium binding protein A9	S100A9	125.0	16.2
Bone morphogenetic protein 2	BMP2	22.5	12.4
Integrin-binding sialoprotein (bone sialoprotein, bone sialoprotein II)	IBSP	9.0	12.3
Alkaline phosphatase, liver/bone/kidney	ALP	2.0	11.9
Distal-less homeobox 5	DLX5	3.8	11.2
Homo sapiens parathyroid hormone receptor 1	PTHR1	7.7	8.3
Bone morphogenetic protein 6	BMP6	13.2	5.5
Homo sapiens jagged 1 (Alagille syndrome)	JAG1	-1.9	5.2
Homo sapiens gap junction protein, alpha 1, 43 kDa (connexin 43)	GJA1	2.8	4.6
Runt-related transcription factor 2, transcript variant 1	RUNX2	2.0	2.2
Sp7 transcription factor, osterix	SP7	2.1	2.1
Distal-less homeobox 3	DLX3	1.9	2.0
Gremlin 1, cysteine knot superfamily, homolog (Xenopus laevis)	GREM1	-10.5	-3.8

^†Fold changes based on mean values (n = 6 Osteocel Plus lots).