Callegari, Andrea (2008) Cardiac tissue engineering: use of a collagen scaffold and bone marrow-derived stem cells in a model of acute and chronic myocardial infarction. [Tesi di dottorato]
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1. Cardiopatch in a model of Acute Myocardial Injury (AMI)
In this research project we tested the possibility of using a new biodegradable collagen-based material in vivo at cardiac level. We chose the rat as animal model and the acute necrotizing injury (ANI) as type of lesion. Wistar rats of 200-250 grams were used. After anaesthesia, animals were intubated and ventilated mechanically with room air. The heart was exposed through a left thoracotomy and a left ventricular acute necrotizing injury (ANI, freeze-thaw procedure) was created by three sequential exposures (60 s each, 20 s of non-freezing interval) of a liquid nitrogen-cooled cryoprobe (a stainless-steel cylinder, 8mm of diameter).
The potential of collagen scaffold (Cardiopatch) for attracting angiogenesis/arteriogenesis was studied in vivo by implantation on a healthy (group 2) or cryoinjured left ventricle (group 3) of rats up to 60 days postinjury times. Blood vessel content and extra-vascular cell infiltration were evaluated within the Cardiopatches, the cryoinjury zones, and the "border zones" of the myocardium facing the cryoinjury zones.
In vitro, this biomaterial supported differentiation of cardiomyocytes, smooth muscle and endothelial cells. When implanted in the peritoneum, the scaffold induced a striking neoangiogenesis but also an innate immune response with an abundant macrophage infiltrate and some foreign body giant cells.
In the heart, cardiopatches were almost completely absorbed in 60 days and became populated by new arterioles and capillaries in both intact and cryoinjured heart (arterioles in cryoinjury vs. intact zone were about 2,3-fold higher; capillaries in cryoinjury vs. intact zone were 1.7-fold higher). In turn, cardiopatches exerted a "trophic" effect on the organizing granulation tissue that emerged from the wound healing process increasing vessel density of this tissue of 2.7-fold for arterioles and 4-fold for capillaries.
Interstitial cells in cardiopatches rarely (<1%) expressed markers of cardiogenic stem cells such as Sca-1- or MDR1, whereas markers of neural crest cells GFAP+/nestin+ cells ranged from 3/30% to 30/70% in cardiopatches placed on intact vs cryoinjured heart, respectively. Myofibroblasts and cardiomyocyte were absent but macrophages largely accommodated in the cardiopatches even after 60 days from implantation. Western blotting of cardiopatches detached from intact/cryoinjured hearts confirmed that endothelial and smooth muscle cells but not cardiomyocyte markers were expressed in the patches.
The porous collagen scaffold was able to evoke a powerful angiogenetic and arteriogenetic response in the intact and cryoinjured hearts, representing an ideal tool for therapeutic angio-arteriogenesis and a potentially useful substrate for stem cell seeding.
2. Cardiopatch in a model of Chronic Myocardial Injury (CMI)
After using the collagen scaffold in a model of AMI, this biomaterial was also applied in a model of Chronic Myocardial Injury (CMI) again in a rat myocardium. In view of a possible clinical application, this model seems closer to the clinical picture of chronic ischemia in human.
The animals were divided in 4 experimental groups:
1. animals that received a chronic necrotizing injury (CNI) at cardiac level
2. animals in which the scaffold was applied to the intact heart;
3. animals in which the scaffold was sutured to a cryoinjured heart;
4. Sham-operated animals.
The animals were sacrificed at time points of 15, 30 and 60 days. After collecting and freezing the organs, cardiac cryosections were obtained for the histological analysis: haematoxylin-eosin, Masson's thrichrome, immunoperoxidase and immunofluorescence.
In detail, four zones were studied in the region of cryoinjury or in the intact heart:
Zone 1: damaged tissue in hearts with CNI;
Zone 2: cardiopatch in intact hearts;
Zone 3: damaged tissue in hearts with CNI and cardiopatch;
Zone 4: cardiopatch in hearts with CNI.
When applied in intact or damaged rat heart, the biomaterial was able to attract a remarkable neovascularization, with the formation of capillaries and arterioles. The scaffold promoted neovascularization in the damaged zone, while this zone was not able to induce a important neovascularization in the cardiopatch any longer, as occurred in the ANI model. The mutual influence between the cardiopatch and the zone with CNI was lacking, probably due to a reduction of angiogenic/arteriogenic growth factors released from the granulation tissue. After 15 days from the application of cardiopatch in hearts with CNI, the rise of blood vessel was significant in zone 3, but it slightly decreased at 30 days. The efficacy of the biomaterial is so debatable in prospective of a long run use. In this contest it would be necessary to use angiogenetic growth factors (such as VEGF) in order to improve the neovascularisation.
The biomaterial was also unable to mobilize resident cardiogenic stem cells: in the cardiopatch no cells with this phenotypic pattern were found.
3. Cardiopatch and Bone Marrow derived Mesenchymal Stem Cells (MSCs) in a model of Chronic Myocardial Injury (CMI)
As these results were encouraging, but not sufficient with regard to a more complex therapeutic use of this the biomaterial, it became essential to test an additional manipulation of the cardiopatch.
The patch applied in a model of CMI was therefore injected with BM-MSCs, a phenotypically well-characterized cell population that is extensively used as a therapy in the myocardial infraction both in animal models and in human trials. In addition, these cells constitutively expressed the GFP (Green Fluorescent Protein), a green fluorescent marker that can be easily tracked after transplantation.
The Wistar rats underwent to CNI and after 30 days were divided into the following groups:
1. animals that received cryoinjury alone;
2. animals with CMI that received via an intra-myocardial route the BM-MSCs alone near the damaged zone;
3. animals in which the patch was attached to the injured myocardium and than injected with medium alone;
4. animals in which the patch was attached to the normal myocardium and the cells injected within it;
5. animals in which the scaffold was implanted in the damaged hearts and subsequently injected with about 4x106 GFP+ BM-MSCs.
The day before the second operation the rats started the treatment with 10mg/Kg/day of Cyclosporine (CsA) until the sacrifice to avoid the risk of rejection.
After 15 days post-injection numerous GFP+ cells were found in the myocardium of animals of group 2 (rats with CMI received GFP+ BM-MSCs alone near the damaged zone), or in the cardiac patch and fibrotic myocardium of animals in groups 4 (patch attached to normal myocardium and the cells injected within it) and 5 (scaffold implanted in damaged hearts and subsequently injected with GFP+ BM-MSCs). This suggests that the cells injected in the patch were able to move from the patch to the myocardium.
Also in this case the material was able to induce neovascularization.
After transplant the BM-MSCs were able to activate differentiation programs and form capillaries, arterioles and cardiomyocytes.
Some of the transplanted GFP+ BM-MSCs were positive for endothelial (CD31) and smooth muscle (SM ?-actin) cell marker and were found in capillaries and arterioles, respectively. In particular, in group 5, most of the BM-MSCs were dispersed into the interstice and several participated in vessel formation both in cardiopatch and in the cryoinjured zone. However, the contribute of GFP+ cells to neoangiogenesis in the cryoinjured zone was very limited.
Two weeks after transplantation some of the engrafted MSCs were stained positive for cardiac troponin T. No presence of cTnT positive cells was detected in the injured myocardium of animals in group 5.
In previous models, the use of the collagen scaffold only in cardiac repair was not sufficient to mobilize resident cardiogenic stem cells locally. The use of BM-MSCs was able to avoid the lack of cells with the phenotypic profile of cardiomyocytes, even if the percentage of conversion to this kind of cell was not big enough to ensure an adequate functional recovery.
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