Type I collagen encapsulated mesenchymal stromal/stem cell (MSC) immunomodulation of monocytes for biofilm-associated Staphylococcus Aureus (S. Aureus) elimination and co-culture viability.

 

 

In surgical proceedings implant devices often produce infections and lacking antibiotics that aid the reconstruction of tissue are scarce. Because surgery is a common procedure, the significance of this study was to inspect whether MSCs, in the presence of type I collagen, can add to macrophage functions that fight phagocytosis by attenuating inflammation, decreasing fibrosis, and promoting the clearance of implant-associated bacteria.  Seven 6 and 12 hour cell culture conditions varied between implant (biofilm)-macrophage, biofilm only, biofilm-MSC-collagen, biofilm-MSC-collagen-macrophage, MSC-PEG Gelatin-biofilm, MSC-adherent-biofilm, and minocycline only. At the conclusion of this project, research findings indicated that type I collagen directly help MSC interactions with autologous macrophages to decrease implant device infections.

 

The schematic diagram shows the effect MSCs have on the healing process of infections

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

  

                              Research Procedures

I. Bacterial Culture

Step 1. Fabrication of Trypticase Soy Agar (TSA) plates

Step 2. Implant (biofilm) inoculation procedures incorporating Tryptic Soy Broth (TSB) and the bacterial growth media incubated at 37°C for 24 hours.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

II. MSC-Collagen Encapsulation

Step 1. Counting of Mesenchymal Stromal/stem cells

Step 2. Fabrication of type I collagen concentration

Step 3.Transferring  of MSCs and type I collagen to well plates, allowing gels to swell overnight.

 

III. Whole Blood Macrophage Isolation

Step 1. Autologous human blood donation

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Step 2. Macrophage (human blood cell) collecting and counting

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

IV. Three way culture of MSCs, Macrophages, and Bacterial Strains

Step 1. Placement of macrophages (human blood cells) and implants (biofilms) in transwells for 6 and 12 hour co-culture settings.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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V. Viability Characterization

Step 1. Microscopic snapshots of macrophage and implant conditions

 

Characterization image:  A) MSC-PEG Gelatin-biofilm 6 hour co-culture composite B) MSC-adherent-biofilm 6 hour co-culture composite C) MSC-type I collagen-biofilm 6 hour co-culture composite. D) MSC-PEG Gelatin-biofilm 12 hour co-culture composite E) MSC-adherent-biofilm 12 hour co-culture composite F) MSC-type I collagen-biofilm 12 hour co-culture composite.

 

 

 

 

 

 

 

 

 

 

 

 

 

Characterization image:  A) biofilm- macrophage-biofilm 6 hour co-culture composite B) biofilm- macrophage-biofilm 12 hour co-culture composite

 

 

 

 

 

 

 

Characterization image:  A) macrophage-biofilm 6 hour co-culture composite B) Macrophage-biofilm 12 hour co-culture composite

 

 

 

 

 

 

 

Characterization images:  A) MSC-PEG Gelatin- macrophage-biofilm 6 hour co-culture composite B) MSC-adherent-macrophage-biofilm 6 hour co-culture composite C) MSC-type I collagen- macrophage-biofilm 6 hour co-culture composite. D) MSC-PEG Gelatin- macrophage-biofilm 12 hour co-culture composite E) MSC-adherent- macrophage-biofilm 12 hour co-culture composite F) MSC-type I collagen- macrophage-biofilm 12 hour co-culture composite G) macrophage-biofilm 6 hour co-culture composite H) macrophage-biofilm 12 hour co-culture composite

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Step 2. Bacterial colony count for comparable and concluding (LogCFU/mL) diagrams of tested controls.