Bone Marrow Mesenchymal Stem Cell Aggregates for Treating Deep Skin Wounds


Cuts, bruises, lacerations, abrasions, of the skin are some of the most common injuries. Deeper wounds that extend into the dermis are more susceptible to chronic inflammation and suffer greatly if they are bumped, knocked, or run into. Such wounds are also more difficult to heal, since a full-thickness cutaneous wound usually damages many different structures and cell lineages. Fortunately, the healing of these structures begins directly after production of the wound. Regeneration is largely orchestrated by growth factors such as Vascular Endothelial Growth Factor and Transforming Growth Factor-β. Since mesenchymal stem cells (MSCs) are a good source of these growth factors, they might be promising candidates for treating full-thickness wounds.

Yan Jin and his colleagues at the Research and Development Center for Tissue Engineering in Shaanxi, China have tested the ability of bone marrow-derived mesenchymal stem cells (BMMSCs) to accelerate the healing of deep skin wounds.

Jin and his colleagues, Yulin An, Wei Wei, Huan Jing, Leigo Ming, and Shiyu Lui used bone marrow from mice that had been genetically engineered to express Green Fluorescent Protein (GFP) in their bone marrow. After isolating mesenchymal stem cells from the bone marrow of these mice, they applied the cells to rats that had suffered full-layer skin cutaneous wounds. However, they tested several different ways of applying these cells to the wound.

In one group of rats, GFP+BMMSCs were grown in cell culture that used a growth medium that contained a steroid drug called dexamethasone and ascorbic acid phosphate. These chemicals caused the BMMSCs to grow in the form of clusters of cells called “cell aggregates” that could be scraped off mechanically and readily transplanted onto the wounds without the need for any scaffold.  These cell aggregates grow as sheets of cells that can act as a kind of patch made from healing MSCs that can be easily and readily applied to a wound or other lesion.  In a second group of rats, the same number of GFP+BMMSCs was topically administered around the wound. In the third group of rats, the BMMSCs were given intravenously through tail vein. All three groups of rats received the same number of BMMSCs. Samples from the bed of the wounds were taken at different time points, and the morphological, histological and molecular characteristics of the wounds were analyzed and compared.

(A) The rim of aggregate curled a little on dish bottom. (B) GFP+BMMSCs in the aggregate gave green fluorescence under 509 nm excitation light. (C) The whole aggregate was scratched off the dish. (D) HE staining revealed a certain thickness of the aggregate with cells in it (Bar = 20 nm). (E) RT-PCR showed that BMMSC aggregate presented significantly higher expression of TGF-β and collagen I but had a similar VEGFα expression with normal cultured cells. (N-C: normal cultured cells; A-C: Aggregate cells; **p < 0.05 is considered statistically different.)
(A) The rim of aggregate curled a little on dish bottom. (B) GFP+BMMSCs in the aggregate gave green fluorescence under 509 nm excitation light. (C) The whole aggregate was scratched off the dish. (D) HE staining revealed a certain thickness of the aggregate with cells in it (Bar = 20 nm). (E) RT-PCR showed that BMMSC aggregate presented significantly higher expression of TGF-β and collagen I but had a similar VEGFα expression with normal cultured cells. (N-C: normal cultured cells; A-C: Aggregate cells; **p < 0.05 is considered statistically different.)

According to the results, the BMMSCs administered in cell-aggregates produced the highest expression of pro-healing genes than the other methods. Also animals treated with the BMMSC cell aggregates also showed better vascularization and more regular dermal collagen deposition than the other two groups of rats.

(A,B) Wound bed size and vascularization state in BMMSC-transplanted rats with each control at 4-week post-operation. Yellow dashed circle outside and the dashed line inside showed the original wound size and the left wound bed respectively; (C) Quantification of wound bed size revealed that rats of C-ag group had the smallest wound bed left at 4W. p1 = 0.000, p2 = 0.001, n = 14; (D) Quantification of capillary number revealed that C-ag group models enjoyed the highest capillary density followed by models of Top-ad group and Int-ad group (p1 = 0.001, p2 = 0.000, p3 = 0.000, n = 15).
(A,B) Wound bed size and vascularization state in BMMSC-transplanted rats with each control at 4-week post-operation. Yellow dashed circle outside and the dashed line inside showed the original wound size and the left wound bed respectively; (C) Quantification of wound bed size revealed that rats of C-ag group had the smallest wound bed left at 4W. p1 = 0.000, p2 = 0.001, n = 14; (D) Quantification of capillary number revealed that C-ag group models enjoyed the highest capillary density followed by models of Top-ad group and Int-ad group (p1 = 0.001, p2 = 0.000, p3 = 0.000, n = 15).
(A) HE staining (top, 10×) of 4-week samples showed epithelialization in three BMMSC transplanted groups while not in all their controls. Masson trichrome staining (bottom, 20×) of dermal layer showed a superior collagen deposition with certain direction and thicker bundle for C-ag group and Top-ad group to that of Int-ad group, while the collagen disposition of control groups samples was short without certain direction; (B) RT-PCR confirmed that the samples of C-ag group and the Top-ad group presented the highest collagen I expression among groups and followed by Int-ad group (p1 > 0.05, p2 < 0.05).
(A) HE staining (top, 10×) of 4-week samples showed epithelialization in three BMMSC transplanted groups while not in all their controls. Masson trichrome staining (bottom, 20×) of dermal layer showed a superior collagen deposition with certain direction and thicker bundle for C-ag group and Top-ad group to that of Int-ad group, while the collagen disposition of control groups samples was short without certain direction; (B) RT-PCR confirmed that the samples of C-ag group and the Top-ad group presented the highest collagen I expression among groups and followed by Int-ad group (p1 > 0.05, p2 < 0.05).

Detection of inflammatory cells as ascertained by immunofluorescence staining of inflammatory cells also revealed that the duration of inflammation in the cell-aggregate-treated group was significantly shorter than the other two groups. These results were corroborated by RT-PCR experiments that measured the expression of pro-inflammatory genes in the wound tissue.

RT-PCR showed the expression profile of inflammatory cytokines TNF-α (A, p1  0.05, p2 > 0.05, p3 < 0.05) and immune-regulating gene iNOS (C, p1 < 0.05, p2 < 0.05). Wound bed tissues of C-ag and Top-ad group expressed lower level of TNF-α and IL-1β, which were significantly higher in Control groups. Tissue of C-ag group expressed highest level of iNOS among groups.
RT-PCR showed the expression profile of inflammatory cytokines TNF-α (A, p1 < 0.05, p2 < 0.05, p3 < 0.01) and IL-1β (B, p1 > 0.05, p2 > 0.05, p3 < 0.05) and immune-regulating gene iNOS (C, p1 < 0.05, p2 < 0.05). Wound bed tissues of C-ag and Top-ad group expressed lower level of TNF-α and IL-1β, which were significantly higher in Control groups. Tissue of C-ag group expressed highest level of iNOS among groups.

In situ immunofluorescence staining also demonstrated higher rates of GFP+-cell engraftment in the rats treated with BMMSC cell-aggregates than the other groups.

(A) Immunofluorescence staining on CD45+ lymphocytes on wound bed samples at 4W (counterstained with Hoechst 33342) (Bar = 50); (B) Quantification of CD45+ cell among groups. CD45+ cell infiltration in Top-ad and Int-ad wound bed tissue was heavier than that that in C-ag ones. p1 = 0.003, p2 = 0.000, p3 = 0.405, n = 6; (C) Immunofluorescence staining on GFP+ cell on wound bed samples at 4W (counterstained with Hoechst 33342) (Bar = 50 nm); (D) Quantification of GFP+ cell among groups indicated better engraftment for C-ag group than the other two cell transplanted groups, difference being significant. p1 = 0.001, p2 = 0.000, p3 = 0.135, n = 6.
(A) Immunofluorescence staining on CD45+ lymphocytes on wound bed samples at 4W (counterstained with Hoechst 33342) (Bar = 50); (B) Quantification of CD45+ cell among groups. CD45+ cell infiltration in Top-ad and Int-ad wound bed tissue was heavier than that that in C-ag ones. p1 = 0.003, p2 = 0.000, p3 = 0.405, n = 6; (C) Immunofluorescence staining on GFP+ cell on wound bed samples at 4W (counterstained with Hoechst 33342) (Bar = 50 nm); (D) Quantification of GFP+ cell among groups indicated better engraftment for C-ag group than the other two cell transplanted groups, difference being significant. p1 = 0.001, p2 = 0.000, p3 = 0.135, n = 6.

These data show that not only are BMMSC cell aggregates safe, but they might also stimulate greater cutaneous regeneration for full layer cutaneous wounds than BMMSCs administered by other means.  These successful studies will hopefully be followed by large animal studies to confirm the expandability and efficacy of this technology in larger animals.

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mburatov

Professor of Biochemistry at Spring Arbor University (SAU) in Spring Arbor, MI. Have been at SAU since 1999. Author of The Stem Cell Epistles. Before that I was a postdoctoral research fellow at the University of Pennsylvania in Philadelphia, PA (1997-1999), and Sussex University, Falmer, UK (1994-1997). I studied Cell and Developmental Biology at UC Irvine (PhD 1994), and Microbiology at UC Davis (MA 1986, BS 1984).