Vascular Endothelial Growth Factor – Releasing Scaffolds Enhance Vascularization and Engraftment of Hepatocytes Transplanted on Liver Lobes

Citation and Paper Link:

Cohen S, Dvir-Ginzberg M, Gamlieli-Bonshtein I, et al. “Vascular Endothelial Growth Factor – Releasing Scaffolds Enhance Vascularization and Engraftment of Hepatocytes Transplanted on Liver Lobes.” Advances in Tissue Engineering: Angiogenesis. 2005 May-Jun; 11(5-6): 715-22.

http://www.ncbi.nlm.nih.gov/pubmed/15998213

Introduction/Motivation

There is currently an interest in developing new treatments for end-stage liver disease and enzyme deficiency by transplanting hepatocytes into affected livers and inducing the regeneration of healthy tissue in vivo. Unfortunately, the two most popular hepatocyte transplantation methods are currently portal vein injection and mesenteric membrane implantation, which have both proven to be unreliable, mainly due to poor cell viability. The extreme vascularization of liver tissue prevents a unique challenge for the engineering of tissue-regenerating scaffolds because implanted cells must have immediate access to oxygen and nutrients in circulation.

The paper by Cohen S, et al, investigates the viability of hepatocytes which have been injected into scaffolds impregnated with VEGF – a known signaling molecule involved with liver regeneration – and implanted into rat livers. The novel approach of this study was to achieve increased cellular engraftment by first inducing the growth of microvessels into the scaffolds before injecting hepatocytes.

Summary of Methods and Results

For the initial stage of the study, two experimental groups were created in order to determine the time dependence of scaffold vascularization: 1) alginate scaffolds implanted into the left liver lobe of rats (control), and 2) alginate scaffolds containing VEGF microspheres implanted into the left liver lobe of rats (experimental). Figure 1 shows one of the implanted scaffolds. Alginate tissue scaffolds were used for this study because of their high porosity (~90%) which would allow ample room for vessel ingrowth and hepatocyte engraftment. To produce the VEGF scaffolds, PLGA microspheres were impregnated with VEGF and incorporated into the scaffolds at the time of fabrication. The microspheres were impregnated with 3.75 μg per 200 mg of PLGA and incorporated into the scaffolds at 40 mg of microspheres per scaffold. The kinetics of VEGF release were measured by ELISA and found to be constant at 8-10 ng/day for two weeks, after an initial burst release of 40%.

Figure 1

Two measurements were made at the time points of 3, 7, and 14 days post-implantation: (Fig 2A) the % cross-sectional area of the scaffold replaced by new vessel ingrowth and (Fig 2B) the capillary density in the scaffold measured in #/mm². All data was collected by harvesting the scaffolds from the rats and taking a histological slice through the approximate middle and staining it with H&E.

Figure 2

Figure 2A shows that there was no statistical difference between the control and experimental groups with regards to the cross-sectional area of the scaffold taken up by new tissue ingrowth. Figure 2B, however, shows that the number of capillaries growing into the scaffolds was affected by VEGF and that the experimental scaffold group had a higher blood vessel density. In order to inject hepatocytes into a pre-vascularized scaffold while still allowing for ample space for engraftment, the 7 day mark was chosen as the optimal injection date.

For the hepatocyte engraftment phase of the study, three experimental groups were used: 1) scaffolds implanted into rats with no VEGF and no hepatocyte injection (sham-operated), 2) scaffolds implanted without VEGF, but hepatocytes were injected on day 7 (control), and 3) scaffolds implanted with VEGF microspheres and hepatocytes were injected also on day 7 (experimental). Histological samples of the scaffolds were taken at time points of 1, 3, 7, and 12 days post-injection. The sham-operated group showed no hepatocytes, indicating that any hepatocyte present in the other groups came from the injection. The histological H&E stains from each time point and experimental group were taken and the cross-sectional area of hepatocytes was computed in μm² per slide, as shown in Figure 3.

Figure 3

For this study hepatocyte engraftment was measured as a function of the cross-sectional area of hepatocytes in the histological slides. Figure 3 shows the decrease in hepatocyte area over time in both the experimental and control groups, however the VEGF was found to have an important effect. The VEGF-impregnated scaffold had higher hepatocyte engraftment at each time point and by day 12 had over a 4 times greater area of intact hepatocytes. This data suggests that the VEGF-induced vascularization served to encourage the attachment of injected hepatocytes.

Overall, this study showed that the vascularization of alginate liver scaffolds can be induced by VEGF and that the enhanced ingrowth of vessels has a positive effect on hepatocyte engraftment.

Shortfalls and Possible Improvements

The main drawback of this study is that it does not directly address its motivation: the restoration of liver function through the transplantation of hepatocytes into a host. In order to investigate liver function, they would have had to remove the scaffolds and analyze the liver-specific gene expression of the engrafted hepatocytes through an experimental method such as PCR. By comparing the gene expression of the implanted cells with normal liver tissue expression, the researchers could have measured how much liver function, if any, was restored. Additionally, using healthy livers in the experiment most likely provided results that would be different than if they had used diseased livers. It is possible that healthy livers are more or less likely to vascularize the scaffolds than diseased livers.

A shortfall of the data analysis was the failure of the researchers to report the vascularization of their scaffolds in comparison with normal anatomical values. Again, it is difficult to assess the efficacy of alginate scaffold vascularization if the normal liver vascularization values are not known. In order to improve the study, I would have proposed taking similar cross-sections of liver tissue from the lobe in which the implant was made and comparing vascularization amounts with the scaffold.

Finally, the measurement of hepatocyte cross-sectional area (which was the primary indicator of cell engraftment) was highly dependent on their injection process which was not rigidly standardized across all of the scaffolds. The cells were injected into the scaffolds at “different sites” in order to have a better distribution of cells, but then it was reported that cell engraftment was greater at the periphery of the scaffolds. If some scaffolds received injections closer to areas of higher engraftment rates than others, then the cross-sectional area alone can not be used as a reliable indicator of cell engraftment. An investigation of cell engraftment as a function of scaffold injection site must be conducted before making conclusions about the effect of VEGF on engraftment.