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Tissue engineering and stem cell transplantation are promising novel therapies for myocardial repair. A major barrier to cell survival after transplantation involves inadequate vascularization. Continuous observation of cardiac tissue engraftment and angiogenesis could help understand these processes and allow for identification of the optimal conditions for these therapeutic interventions. We investigated the ability of a skin-fold chamber model to allow for engraftment of differentiated myocardial tissue in mice. Neonatal atrial and ventricular tissues were implanted in the in vivo chambers. All myocardial implants had a high rate of engraftment (86–95%). Tissue engraftment was preceded by a ‘bleeding phase’ in both the atrial and ventricular implants. This occurred earlier in ventricular compared with atrial implants. Spontaneous contractions were observed after an average of 13 days after implantation in all chambers but occurred earlier in ventricular compared with atrial implants. The host cells surrounded the myocardial implants circumferentially, but have limited infiltration into these grafts. This is the first report of successful ectopic engraftment of differentiated myocardium using a skin-fold chamber. This model is invaluable for real-time observation of early angiogenesis and tissue growth during in vivo myocardial engineering and myocardial regeneration.
Advances in stem cell biology have raised considerable hopes for the development of therapeutic approaches for replacing or regenerating cardiac tissue . Angiogenesis is an active, dynamic process and an important step in heart regeneration. The ability to directly observe and quantify angiogenesis and tissue growth in real time is crucial for understanding the intricate mechanisms of cellular interaction and tissue regeneration. The dorsal skin-fold chamber has been used to study angiogenesis in tissues of various origins [2–6]. This model allows real-time observation of tissue growth and angiogenesis in vivo for up to 5 weeks.
Here, we (i) examine the ability of the dorsal skin-fold chamber to allow for engraftment of myocardial tissue in an ectopic location and (ii) directly observe tissue engraftment and angiogenesis following tissue transplantation. Mouse heart tissues were implanted in the dorsal skin-fold chamber and monitored using intravital microscopy. This is the first study to report a successful ectopic engraftment of myocardial tissue using a dorsal skin-fold chamber for direct in vivo studies of angiogenesis and myocardiogenesis. This model may provide new insights into the mechanisms of cardiac regeneration and replacement therapies.
Balb/c and C57BL/6 mice, 10- to 12-weeks old, body weight 18–22 g, were purchased from Harlan Laboratories (Indianapolis, IN, USA). All animal procedures were reviewed and approved by the University of Wisconsin Institutional Animal Care and Use Committee and performed in accordance with the Guidelines for the Care and Use of Laboratory Animals published by the National Institutes of Health.
The mouse dorsal skin-fold chambers were prepared as described previously [2, 7]. Mice were anaesthetized with 5% isoflurane. The back hair was removed and the skin was gently lifted to form an extended double layer. The double layer of the skin was sandwiched between two symmetrical titanium frames (APJ Trading Co. Inc., Ventura, CA, USA). A 12-mm circular area was removed completely from one of the skin layers and the opening was covered with a glass coverslip incorporated into one of the titanium frames. All surgical procedures were performed under sterile conditions. After surgery, the mice were housed individually and allowed to recover for 1–2 days.
Neonatal pups were anaesthetized with isoflurane in a sealed container and the sternum was cut with iris scissors. The hearts were exposed and explanted. A 2-mm Harris Micro-Punch (Ted Pella, Inc., Redding, CA, USA) was used to obtain pieces of left and right heart tissue of similar sizes. The tissue was cultured in a medium (80% DMEM/F12, 20% FBS) for 72 h before implanted into skin-fold chambers.
Animals were immobilized in polyethylene tubes attached to the stage of a microscope (Leica, Bannockburn, IL, USA). Observations were made with ×1.25, ×5 and ×10 objectives. For vessel contrast enhancement, 0.1 ml of 5% fluorescein isothiocyanate (FITC)-labelled dextran (molecular weight: 150 000) was injected through a tail vein. Observations were captured with an intensified CCD camera (DC300F, Leica) attached to the microscope and serially connected to a computer with the software ImageManager 50 (Leica). To analyse the implanted tissue-beating rate, the video was recorded at 30 frames per second with a camera (Moticam 1000, Motic, Hong Kong, PR China) attached to microscope and a Dell PC. The video was imported into ImageJ (NIH; http://rsbweb.nih.gov/ij/). The myocardial beating area was selected and analysed with Stacks Plot tool. The beating rate was calculated by counting the number of frames/20 beats in the plot and converted into beats per minute.
The mice were sacrificed at the end of the experiment (4–5 weeks after tissue implantation). The engrafted tissues were dissected and embedded in paraffin. Five-micrometre thin sections were stained with anti-mouse troponin T (clone 3D6; Abcam, USA; ab10218), rabbit anti-green fluorescent protein (GFP; Invitrogen, Carlsbad, CA, USA; A11122) and 4’,6-diamidino-2-phenylindole (DAPI; Invitrogen; D3571).
SPSS 13 was used for statistical analysis. Means ± SD were calculated from individual values. One-way ANOVA was used to determine the differences between groups.
A total of 41 skin-fold chambers were used. The animals tolerated the chambers well and the implanted tissues, and showed no sign of discomfort. The chambers were observed daily under bright field microscope.
A total of 22 neonatal atrial tissues and 19 neonatal ventricular tissues were implanted. Ninety-five percent of ventricular tissues and 86% of atrial tissues were successfully engrafted in the dorsal skin-fold chambers (Fig. 1A and B). Tissue implants that failed engraftment remained pale, lacked blood perfusion and did not show any signs of vascularization.
Successful tissue engraftment was preceded by an early profuse ‘bleeding phase’ from both the atrial and ventricular implants. The average time to tissue bleeding after implantation was 6.5 ± 3.1 days for the ventricular tissues and 4.9 ± 2.6 days for atrial tissues (P > 0.05). When matured, neovascular networks were imaged and analysed as described previously [2, 8, 9]. For vessel contrast enhancement, FITC-dextran was injected through a tail vein (Fig. 2). The binary images and their skeletonized mimicries were used to calculate three microvascular parameters: functional vascular density = total vascular length/observation area; vascular area = area occupied by vessels/observation area; mean vessel diameter = vascular area/total vascular length. The average functional vascular density was 32.08 mm−1, the vascular area was 0.608 and the mean vessel diameter was 0.158 mm.
Spontaneously beating ectopic myocardial tissues were obtained in both atrial and ventricular neonatal myocardium implanted in the skin-fold chambers. Spontaneous and synchronized contractions were detected in 70% of the ventricular myocardium and in 40% of the implanted atrial myocardium. The contractions began following tissue bleeding and were confirmed by direct ultrasound examination (Supplementary Videos 1 and and2).2). Spontaneous contractions were observed earlier in ventricular implants compared with atrial ones, 9.7 ± 4.1 vs. 16.3 ± 7.1 days, respectively (P > 0.05). The rate of spontaneous contractions were also higher in the atrial implants compared with ventricular implants (173.8 ± 31.7 vs. 109.6 ± 53.2 bpm; P < 0.05). Once tissues began to beat, they continued to beat with the same beating rate for the duration of the observation period (4–5 weeks).
Wild-type ventricular myocardium was implanted into the skin-fold chamber of a universally expressing GFP mouse C57BL/6-Tg(UBC-GFP)30Scha/J (Jackson Labs, Bar Harbor, ME, USA). The implanted wild-type ventricular tissue into a skin-fold chamber of GFP+ mice displayed an increasing area of fluorescence as early as day 2 after implantation (Fig. 3). The GFP+ area overlying the wild-type ventricular implant increased with time (data not shown). To determine whether the GFP+ cells were infiltrating the engrafted tissue, 5-μm-thick paraffin-embedded sections were stained for troponin T and GFP (Fig. 4). Histological analysis revealed host GFP+ cells mostly surrounded the myocardial implant but infiltrated it to a limited extent only. Multiphoton microscopy showed limited infiltration of individual GFP+ host cells up to a depth of 16 µm in the ventricular graft 17 days after implantation.
The ability to observe and quantify angiogenesis and tissue growth in real time is essential for understanding the intricate mechanisms of cellular interaction and tissue regeneration. 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 . Myocardial tissue could be engineered in larger amount and thickness in vivo using femoral arteriovenous loop enclosed in a subcutaneous chamber . However, direct observation of angiogenesis was not possible in these studies.
Skin-fold chamber preparation is an in vivo model that can support continuous observation and quantification of angiogenesis and tissue growth. We report for the first time successful ectopic engraftment of cardiac tissue of both atrial and ventricular origins in a mouse skin-fold chamber model. The engraftment rate was higher with ventricular compared with atrial implants. We are looking to further understand this difference, but its implications may be important in the selection of myocardial cells for myocardial repair procedures since a ventricular phenotype may have a higher rate of engraftment compared with an atrial one. Neonatal atrial and ventricular tissues become vascularized 3–6 days after implantation. Vascularization was preceded by blood extravasation from the implanted tissue. This phenomenon was not encountered with other tissues implanted in the skin-fold chamber before [2, 5, 7, 12, 13]. This could be explained by the very high capillary density of cardiac muscle and small intercapillary distances [14, 15]. Tissue bleeding may also occur because of the specific organization of the microvascular network in myocardium that allows free drainage of postsinusoidal capillaries directly into the heart chambers on the endocardial surface. The mechanism and significance of this phenomenon is unclear. The myocardial tissue bleeding phase lasts for 24–48 h and was more profuse with ventricular than atrial tissues (data not shown). While the endocardial and epicardial surface areas are similar between the ventricular and atrial tissues, the ventricular cut surface area is higher than the atrial cut surface area because of its higher thickness. The extravasated blood was absorbed over several days as the vascular network matured. Nevertheless, the in vivo microscopic examination of changes within the implanted myocardial tissue was obscured during the bleeding and resorption phase. Quantification of angiogenesis within the engrafted tissue can be performed using previously described methods [2, 8, 9], once the extravasated blood no longer obscures the implant.
Although both atrial and ventricular implants began contracting spontaneously a few days after implantation, fewer atrial implants than ventricular implants regained spontaneous beating. The lack of spontaneous contraction under low-powered in vivo microscope in many atrial tissues could be explained by the presence of a fibrillatory or asystolic state in the implanted atrial tissue that is not detectable by means of in vivo microscopy. 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.  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.
To assess the involvement of host cells in myocardial engraftment, we implanted wild-type ventricular myocardium into the skin-fold chamber of a universally expressing GFP mouse. GFP+ cells could be observed over the myocardial tissue implants early after implantation, but these were found to mostly surround and minimally infiltrate the implants on histology and multiphoton microscopy.
The skin-fold chamber model can support the engraftment of differentiated atrial and ventricular myocardium. This model could serve as a valuable tool for dynamic observation, real-time characterization of early angiogenesis and tissue growth that take place during myocardial engineering as well as myocardial regeneration.
This work was supported by grants from the Department of Surgery of the University of Wisconsin – Madison School of Medicine and Public Health.
Conflict of interest: none declared.