Determining a Timeline for Microvascularized Network Development

Ellen Wasserbauer                                    2009 Summer REU @SLU

Histology fluorescing Beta-Tubulin

Abstract

Engineered soft tissue implants are limited to 2mm thickness due to diffusion limitation of capillaries.  Determining a network development timeline will help us understand how to better engineer tissue.  This experiment uses human umbilical vein endothelial cells (HUVECs) in a collagen matrix to form microvascularized networks.  HUVECs (passage 1) were cultured for five days with samples taken every twenty-four hours.  Histology provided information about protein expression, especially Beta- tubulin, von Willebrand factor (vWF), and Masson’s Trichrome.  Western Blots using determined specific protein expression. DNA assay and Proliferating Cell Nuclear Antigen (PCNA) histology examined cell proliferation.  ApopTAG staining examined the viability of the HUVECs.  Histology and Western Blots indicate Beta-tubulin and vWF are produced by cells assisting in endothelial cell migration, structural support, and collagen binding.  Masson’s Trichrome demonstrated the development of the lumen openings and anastomosis as networks matured at day five.  DNA assay and PCNA suggest that cells focus energy on tube formation and network branching over proliferation. A Microvascularized network development timeline may help efficiency and quality of engineered thick soft tissue.

Introduction

Reconstructive Surgery restores, reconstructs, corrects or improves body structures of misshapen areas due to trauma or disease growth.  There are a few different types of materials commonly used in surgery, one type being synthetically made material, like silicon.  This material is biocompatible and meets the standards put forth, however overtime immunesystem foreign body response can cause degradation and failure of the materials.  Other options for use as a soft tissue are skin grafts or the use of cadaver skin.  Grafts are usually affective in replacing small wound areas.  A drawback of grafting is scaring and the amount of graftable tissue decreases as the area of injury increases.  Cadaver grafts, especially with skin, are the most effective of the previously stated options, however they too have drawbacks.  If a graft is accepted by the patient, it usually will remain viable.  Scaring, discoloring, or rejection overtime can lead to the graft dying.

 Current research is examining a way to engineer an in vitro grown tissue (such as skin or breast tissue) to replace synthetically made tissue or grafts.  Implants have been limited to 2mm thick tissue due to diffusion limitations of capillaries.  Absence of a vascular network causes tissue death due to the lack of oxygen and nutrient delivery in implants.   Researchers are looking at ways to incorporated vascular networks in vitro grown tissue so that when the tissue is implanted into the patient it will be easier for capillaries form the body to attach to those in the new tissue and deliver nutrients throughout the tissue.

The most critical time that determines the viability of an implant is the first few days after implantation.  By studying the development of a capillary network over a short five day period, not only will morphology characteristics of a growing network, but protein expression, cell proliferation, and tissue viability at six different time intervals throughout the experiment will be documented.  This timeline will help researchers further understand how to possibly speed the development and efficiency of these networks.

Results

HUVECs suspended in the collagen matrix formed a network by day three of the experiment, and at day five a much more extensive network was prevalent. Beta-tubulin histology shows that the protein is present in the cell from the start (figure 1) and increases in concentration as the network further develops.  HUVECs also appear to produce vWF as the network matures (figure 2).  Triple Immunohistochemistry indicates a higher concentration of beta-tubulin produced over vWF.  Masson’s Trichrome staining demonstrates lumen openings and the breakdown of collagen by MMP 2 produced by the cells (figure 4).  All histology results are consistent with results from western blots (figure 5) showing necessary proteins present in supernatant samples.  ApopTAG tests for cell viability are inconclusive due to over development using peroxidase (figure 6).  However, HUVECs appear to remain viable through day five as supported in Masson’s staining (figure 4).  PCNA showed that HUVECs were not proliferating over the five day period (figure 7) suggesting that HUVECs focus energy to establishing a network and forming tubes over proliferation.  A DNA assay run to also examine cell proliferation turned out inconclusive due to large degree of variance between data.

Discussion

Engineering thick soft tissue is dependent on the ability to incorporate a microvascular network in implants.  Understanding how a capillary network forms is crucial to this goal.  The preexisting network will assist in the delivery of nutrients to all parts of the tissue.  From this experiment HUVECs produce beta-tubulin over a course of five days.  Beta-tubulin is an intermediate filament used for cell locomotion.  It is responsible for HUVEC migration throughout the collagen tissue and also for structural support.  This protein also assisted in anastomosis allow for the network to mature and become more extensive.  Von Willebrand Factor was also apparent throughout the five day experiment and was produced by the cells.  HUVECs acting in synthetic phenotype secreted vWF to help it establish the network and bind to the collagen.

We confirmed that the network that appears to have formed was indeed a capillary network with open lumens and connecting tubes by using Masson’s Trichrome stain.  This technique showed the progression of network development, starting at initial seed where HUVECs were stilled balled up.  Day 1 they feel around the environment to try and establish them.  At days two and three tubes are forming by cells thinning out and rolling up on them.  The collagen within the forming tube is broken down by a protein MMP2, which appears in the western blot at 70 kD.  Days four and five appear Swiss cheese like and show lumen opening and cross section tube formation.  We believe the cells remained viable throughout the five day experiment not because of the ApopTAG test which appeared to have overdeveloped and stained all cells instead of just dead ones, but because of the distinct growth characteristics seen in the Masson’s staining.   Cell proliferation requires energy from the cell.  PCNA testing shows no proliferation meaning that cells focused their energy on creating tubes and networks.

Conclusion

                 In conclusion this experiment successfully created microvascular tissue that remained viable for five days.  The networks were complete with lumen openings, branches and proper proteins to serve as structural support.  Further testing should be done to reassess and reconfirm the viability of the cells and quantify any possible proliferation present.

Acknowledgments

I would like to thank the National Science Foundation for funding this project and Saint Louis University’s Biomedical Engineering Department and Dr. Rebecca Willits for facilitating it.  I would also like to thank my advisor Dr. Cheryl Miller for guiding me on this project.  Finally thank you to Ms Jasmina Mandzukic and Ms Elizabeth Albert for they’re assistance in histology.

References

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Figure 1. Scale bar equals 20 μm.  Histology of Beta-tubulin over course of 5 days.

Figure 2. Scale bar equals 20 μm.  Histology of von Willebrand Factor over course of 5 days.

Figure 4.  Masson’s Trichrome

Figure 5.  Western Blot using 10-20% Tris-HCl gel

Figure 6. ApopTAG

Figure 7. PCNA