Two Hits for Bone Regeneration in Aged Patients: Vertebral Bone Marrow Clot as a Biological Scaffold and Powerful Source of Mesenchymal Stem Cells

Abstract

Recently, the use of a new formulation of bone marrow aspirate (BMA), the BMA clot, has been described. This product entails a naturally formed clot from the harvested bone marrow, which retains all the BMA components preserved in a matrix biologically molded by the clot. Even though its beneficial effects were demonstrated by some studies, the impact of aging and aging-associated processes on biological properties and the effect of BMA cell-based therapy are currently unknown. The purpose of our study was to compare selected parameters and properties of clotted BMA and BMA-derived mesenchymal stem cells (MSCs) from younger (<45 years) and older (>65 years) female donors. Clotted BMA growth factors (GFs) expression, MSCs morphology and viability, doubling time, surface marker expression, clonogenic potential, three-lineage differentiation, senescence-associated factors, and Klotho synthesis from younger and older donors were analyzed. Results indicated that donor age does not affect tissue-specific BMA clot regenerative properties such as GFs expression and MSCs morphology, viability, doubling time, surface antigens expression, colony-forming units, osteogenic and adipogenic differentiation, and Klotho and senescence-associated gene expression. Only few differences, i.e., increased platelet-derived growth factor-AB (PDGF-AB) synthesis and MSCs Aggrecan (ACAN) expression, were detected in younger donors in comparison with older ones. However, these differences do not interfere with all the other BMA clot biological properties. These results demonstrated that BMA clot can be applied easily, without any sample processing and avoiding potential contamination risks as well as losing cell viability, proliferation, and differentiation ability, for autologous transplantation in aged patients. The vertebral BMA clot showed two successful hits since it works as a biological scaffold and as a powerful source of mesenchymal stem cells, thus representing a novel and advanced therapeutic alternative for the treatment of orthopedic injuries.

Keywords: aging; bone marrow; clot; orthopedic injuries; regenerative medicine.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2022 Salamanna, Contartese, Borsari, Pagani, Barbanti Brodano, Griffoni, Ricci, Gasbarrini and Fini.

Figures

FIGURE 1

Clotted BMA (A) after the remotion from the disposable plastic graduated container and (B) in the culture flask.

FIGURE 2

Representative image of (A,C,E) younger and (B,D,F) older MSCs from clotted BMA. (A,B) MSCs from clotted BMA at 6 days of culture; the presence of some red blood cells, platelets, and leucocytes. Magnification ×20. (C,D) MSCs from clotted BMA at 20 days of culture; the homogenous population of spindle-shaped and plastic-adherent cells. Magnification ×4. (E,F) Live/Dead analyses at 20 days of culture; green staining: viable cells; red staining: dead cells.

FIGURE 3

TGF-β 1, BMP-2, bFGF, VEGF, PDGF-AB, PDGF-CC protein synthesis after 72 h of the culture of BMAs’ clot from younger and older donors. PDGF-AB: *** younger versus older (***p < 0.0005).

FIGURE 4

Population doubling time for MSCs from BMAs clot for younger and older donors.

FIGURE 5

(A) Comparison of MSCs’ surface markers expression CD44, CD90, CD105, and CD73 for BMAs clot from younger and older donors; (B) representative histogram of the flow cytometry analysis (FACS) of the MSC-related CD surface markers in BMAs clot from younger and older donors. (A) and (B) demonstrated high positive expression of CD44, CD90, CD105, and CD73 in both MSCs from BMAs clot from younger and older donors.

FIGURE 6

(A) Comparison of MSCs’ surface markers expression CD31, CD34, CD45 for BMAs clot from younger and older donors; (B) representative histogram of the flow cytometry analysis (FACS) of the MSC-related CD surface markers in BMAs clot from younger and older donors. (A,B) demonstrated negative expression of CD31, CD34, CD45 in both MSCs from BMAs clot from younger and older donors.

FIGURE 7

Colony-forming units (CFUs) count of MSCs from BMAs’ clot from younger and older donors after 10 days of culture in basal medium. The circle chart indicates the number of positive colonies. Representative images of CFUs onto 12-well plates were observed after 10 days of culture.

FIGURE 8

(A) Representative images of osteogenic and adipogenic differentiation of MSCs derived from clotted BMAs from younger donors (left: Alizarin Red S staining, magnification ×10, and Oil red O, magnification ×20; right: LIVE/DEAD fluorescent staining, magnification ×10), (B) from older donors (left: Alizarin Red S staining, magnification ×10 and, Oil red O, magnification ×20; right: LIVE/DEAD fluorescent staining, magnification ×10), and (C) images of chondrogenic differentiation from both younger and older donors (Alcian blue–nuclear fast red staining, magnification ×10) differentiation of MSCs derived from clotted BMAs from both younger and older donors.

FIGURE 9

Gene expression measured by semiquantitative PCR of osteogenic (COL1A1, RUNX2, BGLAP), adipogenic (ADIPO Q, PPARγ), and chondrogenic (SOX9, ACAN, COL2A1) markers comparing the differentiation potential of MSCs derived from clotted BMAs from younger and older donors. ACAN: *** younger versus older (***p < 0.0005).

FIGURE 10

Gene expression of MSCs derived from clotted BMAs from younger and older donors measured by semiquantitative PCR for Klotho and senescence-associated gene expression (IL1β, IL1α, IL6, IL8, CCL4, CXCL2, TNFα, and MCP-1).