Vascularization, by Brittany Forkus

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Vascularization: A Significant Hurdle in Tissue Engineering Success

A recurrent theme throughout this semester in 590B has been in identifying the challenges associated with promoting angiogenesis for the successful vascularization of tissue engineering devices and constructs upon in vivo implantation. I was first introduced to this challenge in the development of my initial wiki presentation regarding the Vacanti ‘Ear on a Mouse’. Following my presentation I was asked why I believed chondrocytes were elected by the researchers as the cell derivative of choice in which to seed the synthetic ‘ear’ to promote cartilage growth. Although I failed in answering this question months ago in front of my entire class of peers, I have been given this opportunity to redeem myself.


In the United States alone, over 10,000 people are currently on waiting lists for prospective organ transplants and only a very small percentage of this number actually receive one. Thus the potential for developing a tissue engineering alterative to the donation of live tissue could have serious clinical implications1. However, several challenges remain before the successful construction of bioartifical organs can be realized:


(1) Adequate cell lines must be identified, isolated, and expanded (1).


(2) Cells must be organized into defined spatial organization (2).


(3) Scaffolding microenvironments must be optimized to promote cell growth and differentiation under in vivo conditions (2).


To date, the majority of clinical success in then ‘engineering’ of organs has been restricted to the development of thin structures that don’t exhibit chaotic vasculature. In in vivo conditions, cells are typically only 100-200um away from oxygen and nutrient supplies. Therefore, when thin, avasculature tissue engineering constructs are developed and implanted into the body the host neovascularization is usually sufficient to meet the nutrient requirements of the organ. Upon scale-up for application such as bioartifical hearts, brains, or livers, vascularization becomes a much more difficult task for these systems are much thicker and metabolically complicated (2). This poses major diffusion limitations in regards to getting necessary nutrients to the cells in the hypoxic centers of these scaffolds that can be centimeters away from a potential supply source (1). The process of angiogenesis is reliant on a tightly regulated cascade of events that include the activation of endothelial cells, organization into immature vessels, association with murals cells, and matrix deposition with maturity1. Numerous strategies and techniques have been tested and evaluated to overcome these barriers, including: therapeutic angiogenesis, ‘Flap Pre-Frabrication’, and pre-supplementing the vasculogenic cells within the construct prior to implantation (1).


Current Approaches to Achieve Vascularization

Approach One: Therapeutic Angiogenesis

Several studies have been performed to evaluate the effect of using growth factors to stimulate angiogenesis (1). In initial studies, researchers have evaluated the efficacy of delivering vascular endothelial growth factor (VEGF) as a recombinant protein encoded within a DNA plasmid or an adenoviral vector (2). Using this technique, patients suffering from severe coronary artery disease have realized significant improvement in angina symptoms and cardiac function (3,4). This method works by inducing the cells of the body to essentially produce their own therapeutic by reading the ‘genetic code’ of the injected plasmid vector. However, additional studies have shown that the delivery of too much VEGF can actually be ineffectual as the maturation of blood vessels is highly dependent on the relative levels of cell types such as macrophages, endothelial cells, and smooth muscle cells surrounding the scaffold (5). VEGF naturally promotes inflammation recruiting the host’s macrophages to the site, which are capable of secreting additional VEGF upon localization. These macrophages also further induce the release of the protein by promoting the release of extracellularly bound VEGF causing an excessive influx of this molecule to the potential angiogenesis site and results in the production of leaky, hemorrhagic vasculature (6). It has been additionally found that the presence of peratinocytes allow the vessels formed in the presence of VEGF to remain stable in the molecules absence (7). These early studies have elucidated the complexity of the involved cell-to-cell interactions and the intricate balance that needs to be maintained for proper functionality.

These studies led to the more complex molecular strategies of using protein ‘cocktails’ to promote vascularizaton in vivo. In particular, one study evaluated the effect of sequentially delivery VEGF followed by platelet-derived growth factor (PGDF-BB) using a controlled-release polymer scaffold. With this approach, mature vascular networks were formed with a sufficient coating of smooth muscles when applied to a hind limb ischemia model. The disadvantage of this approach was that it took the endothelial cells of the host more than two weeks to interact with the scaffold (6). Thus, the high transport barriers make this strategy unfeasible for use in higher order structures.


Approach 2: Flap Pre-Fabrication


The premise of the ‘flap pre-fabrication’ method is to implant the tissue engineered construct into a highly vascularized region in vivo to favor blood vessel growth and then subsequently remove and implante it at the site of the bone defect (1). One prominent study in this field occurred when a 56-year old man who had received tumor surgery eight years earlier was seeking reconstruction of his mandible. The bone defect was greater than 7 cm and the man’s head and neck region had been severely compromised due to previous radiation treatment. To prevent the development of donor-site bone defect, the researchers decided to grow a subtotal replacement mandible inside his latissimus muscle. The goal was to use the muscle vasculature to promote angiogenesis prior to the implantation of the graft to the bone defect location. The researchers used a titanium mesh scaffold that was formed to the shape of the defect and coated with isolate BMP9, bovine collagen type I, and bone marrow to serve as an undifferentiated target for recombinant human BMP7 (see Figure 2). The scaffold was then implanted into the latissimus muscle and can be seen in Figure 3 three weeks post-implantation. The scaffold remained in the muscle for 7 weeks, at which time it was removed and relocated to the region of the bone defect. Figure 4 shows the CT scan of the patient three weeks after his final operation. The patient claims that he can happily eat sausage now and believes it is a nice addition to his previous soup and bean diet. However, this process is highly individualistic, costly, time consuming, and long-term studies have yet to be carried out causing the quest for viable vascularization options to continue (8).



Figure 3.Construct in muscle region 3 weeks post-surgery.
Figure 3.Construct in muscle region 3 weeks post-surgery.



















Figure 4.CT scan of patient 7 weeks after the construct was relocated to bone defect. Reference 8.
Figure 4.CT scan of patient 7 weeks after the construct was relocated to bone defect. Reference 8.















Approach 3: Pre-Vascularization of Scaffold Material

One of the most promising methods for attaining successful in vivo responses is by pre-vascularizing the scaffolding material. Levenberg is a pioneer in this field as he has extensively evaluated the effects of different cell lines on scaffolding materials and their respective differentiation processes. One of his most influential studies in the field pertained to his analysis of the effect of preseeding materials with myoblasts and endothelial cells in the presence and absence of embryonic fibroblasts. In his initial co-culture experiments he used just myoblasts and endothelial cells and found that the endothelial cells spontaneously organized into tubular structures within 3D scaffolds enhancing the vascularization capabilities. However, his most significant finding came with respect to his tri-culture experiments where he added fibroblasts to the mix and found significantly increased vessel density. This supports the hypothesis that smooth muscle cells may not only provide physical support for angiogenesis, but may also release growth factors to promote the process. As can be seen in Figure 4, the addition of embryonic fibroblasts facilitated the stabilization of the vessels over time. The fibroblasts that were seeded in the cultures were stained smooth muscle actin positive suggesting effective differentiation. Levenberg than evaluated the clinical potential of his method by developing a 3D scaffold system and subjected it to several in vivo mammalian systems. The researchers wanted to characterize the survival, differentiation, and vascularization capabilities of the material in the more complex atmosphere. They performed studies where they subcutaneously implanted the scaffold into the backs of immunodepressed mice, into the intramuscular regimes of the quadriceps of nude rats, and replaced an anterior abdominal muscle segment of nude mice with the construct. All three systems continued to differentiate in vivo and took on the desired elongated, multi-nucleated morphology . In efforts to further evaluate the success of the vascularization procedure, the researchers used a luciferase-based imaging system. In this application, the introduced the fluorescent luciferase proteins into the intravenous system to evaluate the resulting fluorescence over time (see Figure 5). This experiment was based on the fact that a highly vascularized region would be indicated by a more intense signal.
























Conclusions

Answer I should have given months ago: The reason the Vacanti brothers used chondrocytes is because these cells are the precursors of cartilage formation. Cartilage is an avascular tissue that doesn't have a high demand for nutrients or oxygen. By using these cells they could avoid the complications necessitated by in vivo vascularization.



References

(1) Van Winckle, Allison. "Vascularization: A Tissue Engineering Roadblock." Stem Cell Network. 16 Aug. 2010. Web.

(2) Jain, Rakesh K., Patrick Au, Josh Tam, Dan G. Duda, and Dai Fukumura. "Engineering Vascularized Tissue." Nature Biotechnology 23.7 (2005): 821-23. Print.

(3) Langer, R.S. & Vacanti, J.P. Sci. Am. 280, 86–89 (1999).

(4) Levenberg, Shumait, Jeroen Rouwkema, Mara MacDonald, and Evan S. Garfein. "Engineering Vascularized Skeletal Muscle Tissue." Nature.com. Nature Publishing Group. Web. 07 May 2012. <http://nature.com/nbt/journal/v23/n7/abs/nbt1109.html>.

(6) Blau, H.M. & Banfi, A. Nat. Med. 7, 532–534 (2001).

(7)Richardson, T.P., Peters, M.C., Ennett, A.B. & Mooney, D.J. Nat. Biotechnol. 19, 1029–1034 (2001).

(8) Yang, J. et al. Nat. Biotechnol. 19, 219–224 (2001).

(9) Warnke, Ph, Ing Springer, J. Wiltfang, Y. Acil, H. Eufinger, M. Wehmöller, Paj Russo, H. Bolte, E. Sherry, E. Behrens, and H. Terheyden. "Growth and Transplantation of a Custom Vascularised Bone Graft in a Man." The Lancet 364.9436 (2004): 766-70. Print.

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