Biomaterial implants mediate autologous stem cell recruitment in mice

Abstract

Autologous stem cells, recognized as the best cells for stem cell therapy, are associated with difficult extraction procedures which often lead to more traumas for the patients and time-consuming laboratory work, which delays their subsequent application. To combat such challenges, it was recently uncovered that, shortly after biomaterial implantation, following the recruitment of inflammatory cells, substantial numbers of mesenchymal stem cells (MSC) and hematopoietic stem cells (HSC) were recruited to the implantation sites. These multipotent MSC could be differentiated into various lineages in vitro. Inflammatory signals may be responsible for the gathering of stem cells, since there is a good relationship between biomaterial-mediated inflammatory responses and stem cell accumulation in vivo. In addition, the treatment with the anti-inflammatory drug dexamethasone substantially reduced the recruitment of both MSC and HSC. The results from this work support that such strategies could be further developed towards localized recruitment and differentiation of progenitor cells. This may permit the future development of autologous stem cell therapies without the need for tedious cell isolation, culture and transplantation.

Figure 1. Characterization of cells in wound fluid aspirates using flow cytometry analyses

In wound fluid aspirates, the majority of cells were inflammatory in nature based on (A) CD45+ and (B) CD11b+ marker expression. The wound fluid also contained some number of (C–D) mesenchymal stem cells (CD73+/CD105+/CD90+/CD45−) along with (E–F) hematopoietic stem cells (Lin-/Sca1+/c-kit+). Plastic adherent cells from such aspirates were treated with various specific differentiation medium. (G) Control untreated cells remained largely undifferentiated. At various time points post-treatment cells were stained with (H) von Kossa for osteogenic (day 10); (I) Oil Red O for adipogenic (day 14) (J) and β-tubulin for neuronal differentiation (day 14). Magnification: 200X; Scale bar = 20 μm.

Figure 2. Cells accumulate in the tissue around different microsphere implants (HPC, PLLA, NIPAm, and PP)

Based on the expression of various markers cells were quantified over a period of 7 days. (A) Histological staining of MSCs (with varying sets of markers including CD105+/CD45−/CD34−/CD56−, Stro1+/CD45−/CD34− and SSEA4+/CD45−) surrounding proinflammatory material PP (top panel) vs. NIPAm (lower panel). All positive markers were tagged with red while negative markers were labeled with red. Arrows indicate the MSCs uniquely expressing red color (signifying positive marker expression). All cells were counterstained with DAPI nucleus stain to aid in cell quantification(B) Histological staining of HSCs (Lin-/Sca1+/c-kit+) adjacent to PP materials (top panel) and NIPAm materials (lower panel). Arrows indicate the HSC expressing Sca-1 and c-kit and lacking expression of lineage markers. Images mag 200X. Scale bar = 50 μm. (C) Quantification of density of MSCs and HSCs surrounding different implants and in untreated normal skin. Estimated densities of MSCs and HSCs surrounding different implants are skin control (MSC= 54.58±12.01, HSC=68.25±9.85 cells/mm²); HPC (MSC=372.5±34.1, HSC=393.4±51.7 cells/mm²); PLLA (MSC= 270.1±85.2, HSC= 262±33.9 cells/mm²); NIPAm (MSC= 60.2±13, HSC=65.4±2.3 cells/mm²); PP (MSC= 215.6±89.7, HSC=297.3±32.2 cells/mm²). Data are mean ± SD. Significance of PLLA, NIPAm, PP vs. HPC; * p< 0.05. (D). Images of recruited X-sight labeled MSC to the subcutaneous area with 1-day PLLA microsphere implants. (E) Trend of MSC recruitment to the subcutaneous microsphere implantation sites based on the localized fluorescence intensities.

Figure 3. Effect of microsphere implant and anti-inflammatory agent treatment on cell recruitment kinetics

Quantitative analysis of (A) CD11b+ inflammatory cell number which increased until around day 4 (~ 35%) after which the percentage declined sharply to ~10% on day 14, (B) MSCs (Stro1+/CD45−/CD34−) and HSCs (Lin-/Sca1+/c-kit+) and (C) proportion of MSCs and HSCs recruited to the implantation sites. For 7-day HPC implants, some of the animals were treated with HPC soaked with anti-inflammatory drug, dexamethasone (DXM). (D) Representative histological images of fibrotic capsular region in untreated control vs. DXM treated mice. (Mag 400 X). (E) Overall cell density in fibrotic capsular region in untreated control vs. DXM treated mice. (F) The localized release of DXM on the recruitment of CD11b+ inflammatory cells and CD45+ immune cells in HPC implant-bearing mice. (G) Numbers of stem cells (both HSC and MSC) in the implant tissue and untreated normal skin control. Images mag 200X. Scale bar = 50 μm Data are mean ± SD. Significance of (HPC+DXM) vs. HPC; * p< 0.05.