Cardiac Tissue Engineering in an In Vivo Vascularized Chamber

Abstract

Background— Cardiac tissue engineering offers the prospect of a novel treatment for acquired or congenital heart defects. We have created vascularized pieces of beating cardiac muscle in the rat that are as thick as the adult rat right ventricle wall. Method and Results— Neonatal rat cardiomyocytes in Matrigel were implanted with an arteriovenous blood vessel loop into a 0.5-mL patented tissue-engineering chamber, located subcutaneously in the groin. Chambers were harvested 1, 4, and 10 weeks after insertion. At 4 and 10 weeks, all constructs that grew in the chambers contracted spontaneously. Immunostaining for α-sarcomeric actin, troponin, and desmin showed that differentiated cardiomyocytes present in tissue at all time points formed a network of interconnected cells within a collagenous extracellular matrix. Constructs at 4 and 10 weeks were extensively vascularized. The maximum thickness of cardiac tissue generated was 1983 μm. Cardiomyocytes increased in size from 1 to 10 weeks and were positive for the proliferation markers Ki67 and PCNA. Connexin-43 stain indicated that gap junctions were present between cardiomyocytes at 4 and 10 weeks. Echocardiograms performed between 4 and 10 weeks showed that the tissue construct contracted spontaneously in vivo. In vitro organ bath experiments showed a typical cardiac muscle length-tension relationship, the ability to be paced from electrical field pulses up to 3 Hz, positive chronotropy to norepinephrine, and positive inotropy in response to calcium. Conclusion— In summary, the use of a vascularized tissue-engineering chamber allowed generation of a spontaneously beating 3-dimensional mass of cardiac tissue from neonatal rat cardiomyocytes. Further development of this vascularized model will increase the potential of cardiac tissue engineering to provide suitable replacement tissues for acquired and congenital defects.

Axial vascularization of a large volume calcium phosphate ceramic bone substitute in the sheep AV loop model

“…The inner volume of 16 cm 3 in our chamber in a cylindrical shape emulates a given volume of tissue that certainly would not be sufficiently nourished by diffusion only, thus approaching clinically relevant dimensions. This is the first study demonstrating de novo formation of axially vascularized connective tissue overcoming the previous limitation to tissue volumes below 1 cm 3 (Bach et al, 2006;Lokmic et al, 2007;Morritt et al, 2007).…”

Cardiac tissue engineering using perfusion bioreactor systems

“…Following implantation in vivo, vascularization is the mechanism for increasing the thickness of viable and functional tissue. Two representative examples include the implantation of stacked monolayer sheets of CM 36 and the use of a vascularized chamber 37 .…”

Intravital microscopy to study myocardial engraftment

“…In vitro engineered neonatal myocardial sheets of thicknesses up to 200 μm survive, become vascularized and electrically coupled with the host myocardial cells when grafted onto the surface of an infarcted heart [10]. Myocardial tissue could be engineered in larger amount and thickness in vivo using femoral arteriovenous loop enclosed in a subcutaneous chamber [11]. However, direct observation of angiogenesis was not possible in these studies.…”

“…The organized atrial tissue contractions had a higher rate than the ventricular one, consistent with the rates observed physiologically in murine hearts. Morritt et al [11] reported that some cardiac tissue constructs that did not spontaneously contract at tissue harvest readily responded when paced. We did not pace these implants externally during the in vivo microscopic observation to preserve the longevity of skin-fold chambers.…”