3.4.1 Evaluation of Decellularization Efficiency and Characterization:
In this study, we have taken a thorough and systematic approach to establish a method to develop a tissue engineered mitral valve scaffold using decellularized porcine mitral valves. While decellularization of organs and other heart valves, including and especially the aortic valve, is not uncommon, very few groups have sought to develop a decellularized scaffold for mitral valves(6,7). This is despite a serious need for an improved valvular replacement. Utilization of this niche microstructure as an inductive scaffold has well established potential, however as mentioned a decellularized scaffold must meet several criteria. As mentioned above, the scaffold must remove cellular and nuclear materials according to standards set in current literature, conserve the extracellular matrix and the corresponding mechanical characteristics of native valve, and stability of the scaffold must be ensured.
Our choice of chemical agents was predicated on an already existing aortic valve decellularization protocol(10). In several pilot studies, we used differing time points and eventually different concentrations of the components of the detergent solutions and increased the concentration of the DNase/RNase solutions described above. Unique to the mitral valve are the four tissue types that one must account for in search for an optimized decellularization that removes xenogeneic cellular and nuclear material while still preserving the ECM. While not definitive, macroscopically there is a marked difference when comparing fresh valves, which have red and brown color to them, with the white and
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sometimes translucent coloring of a decellularized valve. In our histological analysis, we sought to show complete removal of the cellular components. Each histological image shows the cross-section of the leaflet or the chordae tendinae. This is to ensure complete view of the ECM, especially to view the three layers of the mitral valve ECM. Lacking in each of the decellularized samples are the cells from the native valves. Pores are now present where these cells used to reside, these pores could eventually be replaced with seeded cells. To further elucidate cellular removal, an IHC for actin, a common cellular protein was shown to have zero expression in the decellularized scaffolds. As part of the criteria established by our group as well as in literature, removal of nuclear material is also essential for a successful decellularization. The ethidium bromide agarose gel electrophoresis performed showed no presence of DNA in both the leaflets and the chordae (Figure 3.1D). Densitometry showed about a 96% removal of DNA when compared to fresh controls. To further meet the decellularization criteria, more quantitative analysis was required. Therefore, the Nanodrop was utilized to measure DNA quantities in ng/mg of ECM. From this, we showed significant differences between fresh and decellularized samples when comparing DNA quantities. Badylak and his group have established that complete removal of all DNA from a scaffold would be impossible, but that an amount less than 50ng/mg of ECM would be acceptable. Both of our samples for leaflets and chordae meet these criteria (Figure 3.1E).
3.4.2 Preservation of ECM and Basal Lamina Components:
While the elimination of cellular and nuclear material from the scaffold is paramount for the success of the scaffold, preservation of the existing niche ECM is of
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equal importance. The ECM as mentioned is an inductive microstructure that can direct cells to migrate, proliferate, differentiate, or encourage apoptosis and degradation of the scaffold. The mechanotransductive cues delivered to cells via an intact mitral valve microenvironment are extremely significant to the reasoning in choosing this as a scaffold. Simply providing an array of collagen and elastin to seeded cells would inevitably render the scaffold useless and undiscernible as a mitral valve scaffold. Therefore, post- decellularization analysis must evaluate how this process affected the complex collagen and elastin matrix after cellular and nuclear materials were removed. This is true for each tissue type under consideration within the mitral valve apparatus.
Results from several histological stains performed show a preserved mitral valve ECM. Movat’s Pentachrome, which stains for many matrix components, showed a loss of GAGs; however, this is expected after using the detergent solution used in the decellularization protocol. The two main structural proteins, collagen and elastin however remained. As seen in the comparison between the fresh and decellularized leaflet and chordae, the structure of collagen and elastin remain. A VVG stain was done specifically to visualize the elastin in the scaffold. Overall, the quantity and quality of the elastin seems to have remained intact after decellularization. This is important because in degenerative valves, elastin, as well as collagen, are fragmented and could lead to mitral valve prolapse and insufficiency(11). Also pertinent to ECM conservation was retaining the basal lamina
predominantly collagen type IV and laminin. Studies have shown that an intact basal lamina provides a scaffold along which cells can migrate, proliferate and regenerate damaged tissues(12–14). An IHC was performed for both of these basal lamina proteins for
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each tissue type in fresh and decelled samples. As expected, the fresh samples stained positively for collagen type IV and laminin. This was also true for the decellularized samples. Therefore, our scaffolds after undergoing decellularization are not only acellular and lack nuclear material, but we were able to retain basal lamina proteins that are essential for a successful recellularization. Not only did we preserve these proteins, but the overall architecture of the heart valve ECM in both the leaflets and the chordae tendinae.
3.4.3 Evaluation of the Scaffold’s Durability:
The mitral valve conducts a complex mechanical performance with the mitral valve structures and requires the integrity and durability of strong collagen and resilient elastin fibers(15,16). As mentioned, a tissue engineered mitral valve would be expected to operate in a harsh environment of continual and repetitive stresses brought on by the demands of the heart. Therefore, a decellularized scaffold must be able to withstand these forces immediately upon implantation. Translatability of this project depends on this immediate functionality for patients. Accordingly, after evaluating the preservation of the ECM, we tested the durability of the decelled scaffold and the effect PGG treatment had on them. PGG, used before for the stabilization of aortic valve scaffolds, was used here for the first time for the stabilization of the complex and heterogeneous structure of the mitral valve. Biaxial mechanical testing was performed on three groups, fresh, decell, and PGG-treated decelled scaffolds (Figure 3.3A, B). From this, we can see that the decellularization process does in fact decrease the strength of the tissue when compared to the fresh samples. Treatment with PGG increased the mechanical strength of the scaffolds in both the circumferential and radial direction slightly higher than native valves. PGG also increased
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the modulus for the chordae tendinae in comparison to the untreated-decelled chordae. Weak and enlarged “floppy” leaflets are often associated with chordal elongation, thinning, and/or rupture. Therefore, chordal modulus is important for the valve’s overall coaptation. Differential scanning calorimetry (DSC) was used to evaluate crosslinking within the scaffolds when compared amongst fresh, decelled, and PGG-treated samples. PGG-treated samples yielded a significantly higher thermal denaturation temperature when compared to the decelled group. This is related to the preservation of the integrity of the collagen and elastin fibers. An evaluation of the scaffold’s durability must also consider withstanding a host response from the patient. Most of the natural and untreated tissues used in scaffolds elicit a host response and are prone to degradation post-implantation(17). Because the mitral must undergo large and repetitious forces during a patient’s lifetime, the scaffold must be able to withstand degradation, degenerative pathologies will ensue, or the construct will fail altogether. In either case, failure of valvular coaptation would persist and the multiple mechanisms in the mitral apparatus would fail to operate properly. Treatment with PGG has shown to circumvent these issues. We evaluated (Figure 3.4A, B) the effectiveness PGG would have on degradation of scaffolds treated with the proteases collagenase and elastase. Fresh, decelled, and PGG-treated acellular scaffolds were compared at both 24 and 48-hour treatments. Remarkably, PGG-treatment significantly increased resistance to degeneration of the scaffolds at both time points. The untreated decelled scaffolds were the most prone to degeneration when exposed to collagenase, and the fresh tissues were the most prone to degeneration when treated with elastase. It appears the decellularization process does affect a scaffold’s degeneration potential and perhaps affects the structural
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proteins collagen and elastin differently, as evidenced the differences in degradation. The increased resistance of PGG-treated scaffolds to the activity of proteases confirms the integrity of these macromolecules. These results may be significant as it was demonstrated that fragmented elastin is prone to calcify in cardiovascular tissues, such as the aortic wall(18). In fact, varying degrees of calcification are found in the mitral valve annulus of degenerating valves. PGG in previous studies has shown to significantly decrease calcification of implanted tissues, and while this was not tested with the present study, we anticipate that the tissue-engineered mitral valve would be protected from in vivo calcification(9).
Besides optimal physical properties and durability, the collagen and elastin-based scaffolds proved to be cytocompatible. The paradigm with which our lab operates advocates for recellularization of the scaffold and construct pre-conditioning in a bioreactor. Therefore, human adipose derived stem cells were seeded into and on the PGG- treated scaffold to assess their potential to survive (Figure 3.5). Live/Dead images as well and nuclear staining showed that not only can cells thrive on the scaffold, but we also have the capacity to seed the scaffold interstitially. This was imperative to the further aims presented. In addition to their survival, the cells appeared to synthesize a layer of GAGs, which we interpreted as constructive remodeling of the leaflets (Figure 3.5E). This cell- matrix interaction is the key to the success of this scaffold. The positive remodeling observed by the initial cell seeding we believe is a result of the well-preserved biochemical composition of the scaffolds, as well as its architecture.