HFF-1 cells prefer to grow in lower concentration of Fibrinogen. Higher concentration ECMs could be too dense for cell proliferation, restricting cell migration and spreading. Increasing the concentration of Thrombin helped the cells to proliferate after eight days. This could be due to a change in the physical nature of the construct, allowing cells to migrate and proliferate. A plateau was not reached during this timeframe, and the experiment should be continued for a longer duration (14 days). Reduction in proliferation on Day 8 for the Fib5/Th5 construct could be due to either space restriction or disintegration of the construct. Increasing the concentration of Fibrinogen might result in a different density of the construct and further investigation should be done on the stiffness of the ECM. mMSCs proliferated best on Day 8. This can be explained in terms of the adaptation of cells in a new type of construct. The cells were grown in a 2D setup as inoculant and, when transferred to a 3D construct, were learning to migrate in a different type of environment. The highest growth in Fib5/Th10 construct on Day 8 was in accordance with HFF-1 cell results as well. The physical change in the construct could be responsible for this increased proliferation. Changing the concentration of either Fibrinogen or Thrombin independently did not affect the cell growth, but the growth was affected by how these two interacted to make the final construct.

Human Foreskin Fibroblast and Mouse Mesenchymal Stem Cell Human Foreskin Fibroblast and Mouse Mesenchymal Stem Cell BehaviorBehavior

in 3D Fibrin Constructsin 3D Fibrin ConstructsAlvarado, R* ,Burke, K*, Dalton, J*, Sidhu, R*, Soto, R*, McCoy, M • Tawil, B, PhD, Instructor • Biomedical Engineering 501

California State University Channel Islands • One University Drive • Camarillo, CA 93012

300 µL fibrin gel cultures were created with human fibrinogen (Sigma) solubilized in 0.85% sodium chloride at 37°C and thrombin (Sigma) reconstituted in 1 mL of sterile dH2O. HFF-1s (ATCC) and mMSCs (Invitrogen) were mixed with fibrin at 1,000,000 cells/mL (50,000 cells/well) in 24-well plates, a cell density found to maximize proliferation (cell concentrations were calculated using measurements obtained from the T4 Cellometer [Nexcelom]) and thrombin was added immediately following. Final fibrinogen concentrations were 5 and 10 mg/mL. Final thrombin concentrations were 5 and 10 IU/mL. Non-coated control wells were also measured. 3D fibrin constructs were incubated at 37°C with 15% serum-containing DMEM; media was changed at the Day 4 timepoint in Day 8 plates. Measurements were taken one, four, and eight days after seeding, where day one samples were processed an hour after fibrin construct formation. HFF-1 and mMSC proliferation was evaluated by labeling cells with Calcein AM (Invitrogen) and imaging the cells using an IX-71 inverted microscope (Olympus) and Qcapture pro software (QImaging) as well as reading average fluorescence intensity (494 nm ex / 517 nm em) over each well. FilterMax F3 Multi-mode Plate Reader Software (Molecular Devices) provided raw fluorescent read data. MiniTab 16 software was used to evaluate raw data produced from the plate reader and produce graphical results including standard deviations.

Materials and Methods

The generation of a fibrin scaffold during wound healing is considered the normal healing process and typically precedes scab formation. In order to achieve proper scaffold formation, this process requires a combination of many constituents including fibrinogen, and thrombin, in coordination with extracellular components such as collagen, growth factors and white blood cells. Furthermore, the binding of proteases, among other factors, is responsible for facilitating scab development. Successful healing is contingent upon an adequate balance of all constituents as well as appropriate environmental conditions. The purpose of this experiment was to achieve in vitro growth of two cell types, namely Human Foreskin Fibroblasts (HFF) and mouse Mesenchymal Stem Cells (mMSC), within fibrinogen / thrombin scaffolds. More specifically, these scaffolds were created at varying fibrinogen and thrombin concentrations and subsequently assessed for cell proliferation.

Introduction

AbstractDuring the proliferative phase of the wound healing process, a fibrin scaffold is formed, allowing the formation of a scab. This allows fibroblasts to grow and secrete a new extracellular matrix (ECM), thus allowing new epithelial cells to populate the area and form new tissue. This process occurs through a series of complex biochemical events using thrombin, fibrinogen, and other constituents at very specific concentrations. In our experiment, we grew mouse Mesenchymal Stem Cells (mMSCs) and Human Foreskin Fibroblast Cells (HFF) within a 3 dimensional fibrinogen/thrombin scaffold using varied concentrations of each constituent. The cells were fluorescently stained and imaged with a fluorescent microscope at the following time points: Day 0, 4, and 8. Our results show that HFF cells preferentially grow in low concentrations of fibrinogen and that mMSCs showed a slight preference for low concentrations of fibrinogen, although, not as drastic as the HFF cells. We also found that there was a large increase in proliferation of HFF cells between day 0 and day 4, whereas the large increase in proliferation amongst mMSCs occurred between day 4 and 8.

Figure 1, Proliferation of HFF-1 cells: Cell proliferation was highest at Fibrinogen 5mg/mL with 5IU/mL of Thrombin on Day 4. Proliferation was slower in Fib5/Th10 treatment on Day 4, but there was a sharp increase observed on Day 8, making it the highest for Day 8. Cell proliferation was lowest in Fib10/Th5 treatment both on Day 4 and Day 8. This was even lower than the non-coated control. Increasing the concentration of Fibrinogen while keeping the concentration of Thrombin constant decreased cell proliferation.

Figure 2, Fluorescent micrographs of HFF-1 cells

Figure 3, Proliferation of mMSCs: Cell proliferation was highest on Day 8 for all constructs, including the non-coated control. Increasing the concentration of Thrombin increased the proliferation for Fibrinogen concentration of 5mg/mL. Cell proliferation was highest in Fib5/Th10 constructs both on Day 4 and Day 8. The cells proliferated at equal or better rates in the non-coated control on Day 4.

Figure 4, Fluorescent micrographs of mMSCs

* = equal contributor

Results

Conclusions