Use of Autologous Auricular Chondrocytes <br/>For Lining Left Ventricular Assist Devices

Use of Autologous Auricular Chondrocytes For Lining Left Ventricular Assist Devices Doreen Rosenstrauch228291.5672.2.1.2.1.1.8570#2242.1.2.92.1.3.102.1.4.2.1.5.112.1.6.12132.1.7.14510#3792.2.152.2.1.2.2.2.2.2.3.162.2.3.1.2.2.3.2.172.2.4.2.2.5.183.193.1.3.1.1.274#2203.1.2.20536#3633.1.3.21536#3713.1.4.22528#3263.2.233.2.1.539#2373.2.2.24547#26225532#2654.2627 REFERENCES3031 ACKNOWLEDGMENT32enTable of contentsHelp Aus der Klinik
für Herz-, Thorax- und Gefäßchirurgie des Deutschen Herzzentrums Berlin
der Medizinischen Fakultaet Charité
der Humboldt- Universitaet zu Berlin
Berlin, Germany

und

aus dem
Texas Heart Institute at St. Luke's Episcopal Hospital/
The University of Texas Health Science Center at Houston
Texas, USADISSERTATIONUse of Autologous Auricular Chondrocytes <br/>For Lining Left Ventricular Assist Devices Zur Erlangung des akademischen Grades
Doctor medicinae (Dr. med.)Vorgelegt der Medizinischen Fakultaet Charité
der Humboldt- Universitaet zu Berlinvon

Doreen Rosenstrauch

aus Zossen

Prof. Dr. Joachim W. Dudenhausen Prof. Dr.med. Hans H. Scheld PD Dr.med. Michael Settinger Prof. Dr.med. Roland Hetzer Datum der Promotion: 23. April 2004 ABSTRACT

Background: Auricular elastic cartilage is a potential source of autologous cells for lining the luminal surfaces of left ventricular assist devices (LVAD’s) to improve long-term biocompatibility. We evaluated this potential in vitro and in vivo in a calf model.

Methods: In vitro, auricular cartilage was harvested from the anesthetized ear of a calf, isolated, and cultured on tissue culture dishes. Primary chondrocytes were typed by immunocytochemistry, transferred into culture media, passaged twice, and seeded onto the blood-contacting luminal surfaces of four LVAD’s (HeartMate®; Thermo Cardiosystems, Inc., Woburn, MA). The seeded cell linings were preconditioned under flow conditions in vitro to promote cell adhesion to the luminal surfaces. Seeding efficiency and cumulative cell loss under flow conditions in vitro were quantitated. In vivo, one of the four preconditioned, autologous chondrocyte-lined LVAD’s was implanted into the tissue-donor calf; run for 7 days; explanted; and finally evaluated grossly, by scanning electron microscopy, and by transmission electron microscopy.

Results: Autologous chondrocytes were seeded onto the luminal surfaces of the four LVAD’s. The seeding efficiency was 95.11± 4.23% (n = 4). Cumulative cell loss during preconditioning under flow conditions in vitro did not exceed 12% (n = 4). After 7 days of in vivo implantation, the luminal surfaces of the implanted LVAD demonstrated an intact, strongly adherent cellular lining. There was no evidence of thromboembolic events at necropsy.

Conclusions: Auricular elastic cartilage is a ready and easily accessible source of chondrocytes whose ability to produce collagen II and other important extracellular matrix constituents allows them to adhere strongly to the luminal surfaces of LVAD’s. The simple method of isolating and expanding auricular chondrocytes presented here could be used to provide autologous cell linings for LVAD’s and other cardiovascular devices to improve their long-term biocompatibility. Our successful short-term feasibility study in a calf model warrants further study in vivo.

auricular chondrocytes tissue engineering left ventricular assist device elastic cartilage Table of contents
1. INTRODUCTION
2. MATERIALS AND METHODS
2.1. In Vitro Experiments
2.1.1. Tissue Harvesting
2.1.2. Tissue Isolation
2.1.3. Cell Culture
2.1.4. Histology
2.1.5. Immunocytochemistry
2.1.6. Cell Seeding
2.1.7. Cell Preconditioning
2.2. In Vivo Experiments
2.2.1. Animal Care
2.2.2. LVAD Preparation
2.2.3. LVAD Implantation
2.2.3.1. Operative Procedure.
2.2.3.2. Postoperative Care.
2.2.4. Necropsy and Gross Observations
2.2.5. Scanning Electron Microscopy and Transmission Electron
3. RESULTS
3.1. In Vitro Experiments
3.1.1. Histology
3.1.2. Immunocytochemistry
3.1.3. Seeding Efficiency
3.1.4. Cumulative Cell Loss During Preconditioning
3.2. .In Vivo Experiments
3.2.1. Necropsy and Gross Observations
3.2.2. SEM and TEM
4. DISCUSSION
REFERENCES
ACKNOWLEDGMENT
Tables
Table 1. Composition of Modified RPMI 1640 Cell Culture Medium
Table 2. Composition of 2% Antimycotic-Antibiotic Phosphate-Buffered Saline Solution
Images
FIGURE 1 Harvest of bovine auricular cartilage from the ear of the tissue-donor calf. (Left) A 2-mm-diameter biopsy sample (arrow) of auricular cartilage being held by a pickup. The trephine used to obtain the sample is shown still in the ear. (Right) Biopsy site (arrow) at the ear after the trephine was removed.
FIGURE 2 In vitro flow loop used for preconditioning of seeded cells. The loop consisted of a reservoir filled with cell culture medium (1) and the cell-seeded LVAD (2). Samples of cell culture medium emptied into the reservoir via a stop cock (3) were collected from the reservoir at several time points during the 12-hour preconditioning period as described in Materials and Methods. The in vitro flow loop was connected to a pneumatic drive console (not shown).
FIGURE 3 Chondrocytes (Cc) growing from a piece of elastic cartilage (Ec) at zero passage. Tissue culture cells (Cc) derived from isolated auricular cartilage tissue (Ec) at zero passage stained positive for elastic and collagen fibers, indicating the presence of chondrocytes.
FIGURE 4 Immunocytochemistry of elastic cartilage (left) and chondrocytes in tissue culture of second passage (right). Positive staining for collagen II indicated the presence of chondrocytes.
FIGURE 5 Seeding efficiency. The first seeding efficiency of four LVAD’s was 92.66 ± 10.08% and the second seeding efficiency of four LVAD’s was 98.14 ± 0.93%. Total seeding efficiency of four LVAD’s was 95.11 ± 4.23%.
FIGURE 6 Cumulative cell loss (n = 4). The cumulative cell loss during preconditioning under flow conditions on the in vitro flow loop was 2.22 ± 0.32% after 30 minutes, 5.13 ± 0.15% after 3 hours, 7.47 ± 0.20% after 5 hours, 9.78 ± 0.35% after 7 hours and 11.45 ± 0.21 after 9 hours.
FIGURE 7 Gross appearance of the implanted LVAD’s biomaterial surfaces after 7 days of implantation in vivo. (Left) Textured polyurethane surface; (right) sintered titanium surface. The luminal surfaces of the LVAD are completely covered with an intact cell layer.
FIGURE 8 Scanning electron microscopy of the implanted LVAD’s biomaterial surfaces after 7 days of implantation in vivo . An extensive amount of extracellular matrix and an intact, well-incorporated cellular coating (arrows) were noted on the textured polyurethane (left) and sintered titanium (right) surfaces of the implanted LVAD.
FIGURE 9 Transmission electron microscopy of the implanted LVAD’s biomaterial surfaces after 7 days of implantation in vivo . (Left) Textured polyurethane surface; (right) sintered titanium surface. A well-established monolayer of chondrocytes was revealed. No endothelial cells were seen.