A method for seeding titanium blood-contacting biomaterials with autologous cells and testing biocompatibility is described. This method uses endothelial progenitor cells and titanium tubes, seeded within minutes of surgical implantation into porcine venae cavae. This technique is adaptable to many other implantable biomedical devices.
Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk of thromboemboli formation1,2,3. We have developed a method to line the inside surface of Ti tubes with autologous blood-derived human or porcine endothelial progenitor cells (EPCs)4. By implanting Ti tubes containing a confluent layer of porcine EPCs in the inferior vena cava (IVC) of pigs, we tested the improved biocompatibility of the cell-seeded surface in the prothrombotic environment of a large animal model and compared it to unmodified bare metal surfaces5,6,7 (Figure 1). This method can be used to endothelialize devices within minutes of implantation and test their antithrombotic function in vivo.
Peripheral blood was obtained from 50 kg Yorkshire swine and its mononuclear cell fraction cultured to isolate EPCs4,8. Ti tubes (9.4 mm ID) were pre-cut into three 4.5 cm longitudinal sections and reassembled with heat-shrink tubing. A seeding device was built, which allows for slow rotation of the Ti tubes.
We performed a laparotomy on the pigs and externalized the intestine and urinary bladder. Sharp and blunt dissection was used to skeletonize the IVC from its bifurcation distal to the right renal artery proximal. The Ti tubes were then filled with fluorescently-labeled autologous EPC suspension and rotated at 10 RPH x 30 min to achieve uniform cell-coating9. After administration of 100 USP/ kg heparin, both ends of the IVC and a lumbar vein were clamped. A 4 cm veinotomy was performed and the device inserted and filled with phosphate-buffered saline. As the veinotomy was closed with a 4-0 Prolene running suture, one clamp was removed to de-air the IVC. At the end of the procedure, the fascia was approximated with 0-PDS (polydioxanone suture), the subcutaneous space closed with 2-0 Vicryl and the skin stapled closed.
After 3 – 21 days, pigs were euthanized, the device explanted en-block and fixed. The Ti tubes were disassembled and the inner surfaces imaged with a fluorescent microscope.
We found that the bare metal Ti tubes fully occluded whereas the EPC-seeded tubes remained patent. Further, we were able to demonstrate a confluent layer of EPCs on the inside blood-contacting surface.
Concluding, our technology can be used to endothelialize Ti tubes within minutes of implantation with autologous EPCs to prevent thrombosis of the device. Our surgical method allows for testing the improved biocompatibility of such modified devices with minimal blood loss and EPC-seeded surface disruption.
1. Endothelial progenitor cell isolation
2. Titanium tube assembly
3. Seeding device and component assembly
4. EPC-seeding of Ti tube inner surface
5. Implantation of Ti tube into porcine inferior vena cava
6. Explantation of Ti tube
7. Fixation and imaging Ti inner surface
8. Representative results:
Following execution of this protocol, physicians and scientists are able to endothelialize solid tube structures with autologous blood-derived endothelial progenitor cells in a large animal model. Figure 2 shows that EPCs isolated with our method appear as colonies with cobblestone morphology after approximately 7 days in culture. Our seeding device illustrated in Figures 5 and 6 allows for slow rotation of Ti tubes filled with the EPC suspension and results in uniform coverage of the tube’s inner surface under sterile conditions9.
Our implantation surgery allows for testing the propensity of biomaterials, such as Ti, for thrombosis in a large animal model. We found that pigs tolerate this procedure well and that this implantation can be achieved with only minimal blood loss and without EPC layer disruption.
Figure 7 shows that a bare Ti tube completely occludes, whereas our EPC-lined tube remains patent even in the prothrombotic low shear environment of the inferior vena cava. Further, the presence of a confluent layer of fluorescently-labeled cells confirms the success of this method as shown in Figure 8.
Figure 1. Schematic of autologous endothelial progenitor cell (EPC) seeding experiment. First, peripheral blood is drawn from a pig. Next, EPCs are isolated from the blood and expanded in culture. EPCs are then used to line a titanium (Ti) tube device, which is then surgically implanted into the inferior vena cava of the same pig from which cells were isolated.
Figure 2. Representative colony of EPCs in culture, approximately 7 days following isolation procedure (imaged with an inverted Leica DMIL microscope with Imaging camera and QCapture software).
Figure 3. Ti tube sections prior to assembly. Ti tubing is cut into 3 equal sections longitudinally, and then cut to 4.5 cm length. Inner surfaces of Ti sections are polished using a bench grinder and emery cloth to remove visible pits.
Figure 4. Assembly of Ti tube sections with PVC heat-shrink tubing (blue) and heat gun. Ti sections are supported on a machined aluminum mandrel which extends through Ti sections and matches the dimensions of the Ti tube inner diameter.
Figure 5. Titanium tube assembly, showing all components: luer cap, syringe ‘head piece,’ silicone tubing, and Ti tube with PVC wrap (blue). Assembly is put together prior to sterilization for surgical use.
Figure 6. Ti tube seeding setup inside incubator, showing motor, platform, machined aluminum syringe holder, Ti tube assembly, and 5 cc syringe. Note: Assembly is shown without protective sterile sheath for visualization purposes.
Figure 7. Representative gross results of implantation surgery. (A) End-view of control bare metal Ti tube after implantation in porcine inferior vena cava (IVC). Tube lumen is fully occluded with a solid clot. (B) End-view of EPC-seeded Ti tube after implantation. Tube lumen is fully patent and clear. (C) Dissected view of control (bare) Ti tube after 3 day implantation, showing extent of thrombosis (experiments were conducted up to 3 weeks duration with identical results).
Figure 8. EPCs on Ti tube surface following 3 day implantation (imaged with an upright Leica DMRB microscope with a QImaging QICAM monochrome digital camera and Image Pro Plus software). (A) Confluent cells on surface showing PKH26 pre-surgery labeling. (B) Confluent layer of EPCs. Red: PKH26 pre-surgery labeling. Green: EPC PECAM-stain.
The method of cell-seeding Ti tubes presented here enables physicians and scientists to quickly and uniformly endothelialize blood-contacting surfaces of implantable devices. Since we isolate and expand EPCs from peripheral blood samples, no major invasive procedure is required to harvest these cells. Moreover, the EPCs are autologous; therefore, the risk of any immune reaction to the cell-seeded implant is eliminated. The principles demonstrated in his protocol can be utilized not only for Ti tubes, but for many other biomaterials, which are utilized in cardiovascular medicine.
Critical steps in this protocol are meticulous cleaning of the Ti tubes, as we found that any adherent film of contaminant compromises cell adherence. Further, a slow rotation speed (inversely proportional to the tube diameter) is essential during the seeding process, such that EPCs can slowly settle and adhere to the surface as the Ti tube is moving.
Our method of seeding immediately before implantation avoids impractical ex vivo culture times; cells adhere individually and later form a confluent sheet in vivo, avoiding the possibility of embolization as a sheet immediately following the re-establishment of flow. Our prior studies show that once the EPCs have grown to a confluent layer, they make an extra-cellular matrix to which they firmly adhere, additionally minimizing any possible sloughing of an endothelial sheet. Although the possibility of embolic detachment cannot be entirely ruled out, it seems to be multifold lower risk than thrombosis of the entire device surface, the problem that this therapy is designed to prevent.
Our approach of implantation into the low shear, prothrombotic environment of the inferior vena cava utilizes one of the most trusted large animal models for researching blood compatibility and thrombosis of devices5,6. Note that all animal care and experimentation was conducted in accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals and only after approval of the Duke University Institutional Animal Care and Use Committee.
In order to successfully utilize this implantation method to test biomaterials and devices, it is important to patiently skeletonize the IVC segment and ligate all venous branch vessels in preparation for device insertion, such that no bleeding around the device originates in a ‘false lumen’. Another critical step is the addition of DPBS into the device lumen such that the cells on the inside tube surface remain moist during closure of the veinotomy and before reperfusion is initiated. If the device cannot be found in the location where it was implanted, it may have migrated ‘upstream’ in the IVC. This can be prevented by placing a suture (4-O Prolene) through the vein wall and through a 2 – 3 mm section of the PVC tubing so that the tube is firmly anchored in its present location. Should the researcher have difficulty finding the fluorescently pre-labeled cells shown in Figure 8 after explantation in an otherwise patent tube, it is likely that the cells have peeled off as a coherent sheet. This can be prevented by very gentle dissection of the surrounding vein and dis-assembly of the 3 Ti tube sections, after fixing the vein together with the tube.
Our technology provides proof-of-concept for preventing cardiovascular device thrombosis through EPC-seeding. This technology may be used in the development of ‘biogenic’ implants lined with patients’ own endothelial progenitor cells. The feasibility study in our porcine animal model provides for the first steps toward translation of this ‘personalized medicine’ into clinical practice.
The authors have nothing to disclose.
The authors would like to thank Leica Microsystems for their valuable advice on imaging titanium sections and Gemini Bio-Products for providing the porcine serum used in this study. We also like to thank the NIH for their support through Grant “Autologous EPC lining to improve biocompatibility of circulatory assist devices”, RC1HL099863-01. Further, we are grateful for the National Science Foundation Graduate Research Fellowship’s support of Alexandra Jantzen. We also like to thank George Quick, Mike Lowe and Ianthia Parker for assisting with many aspects of the surgical procedure and handling of our research animals. Steven Owen has been invaluable in machining many parts of our seeding device and cutting titanium tubes.
Reagent | Company Name | Catalogue Number | Comments |
Acepromazine | Boehringer-Ingelheim | BIC670025 | NAC# 10280002 |
Alconox Powered Precision Cleaner | Alconox | 1104 | |
Balfour Surgical Retractor | Adler | N/A | |
Baytril | Bayer | APVMA 46028/0705 | |
Butterfly Needle (19G) | Terumo | SV19CLK | |
Chlorhexidine | 3M | 9200 | |
Clamps (45-degree) | Aesculap | FC339T | |
DPBS (-/-) | Gibco | 14190-144 | |
DPBS (+/+) | Gibco | 14040-133 | |
DuraPrep | 3M | 8635 | |
EBM-2 Medium | Lonza | CC-3156 | Base for both serum free and full growth medium |
EGM-2 SingleQuots | Lonza | CC-3162 | Used with EBM-2 for both serum free and full growth medium |
Electrocautery Tool | Valleylab | SurgII-20 | |
Emery Cloth | 3M | 60-0700-0425-8 | 240 grit |
Euthasol Euthanasia Solution | Virback Animal Health | ANADA #: 200-071 | |
Fentanyl Patch | Actavis | NDC # 67767-120-18 | |
Flunixin | Schering-Plough | NAC #: 10470183 | |
Foley Catheter (16F) | Bard | 730116 | |
HBSS | Sigma | H8264-500ML | |
Heat Gun | Milwaukee | 8988-20 | |
Heparin | NDC #: 25021 | ||
Histopaque-1077 | Sigma | H8889-500ML | |
Intubation Tube | Mallinckrodt | 86113 | |
Isoflurane | MWI, Meridian | NDC #13985-030 | |
IV Catheter (18G) | Becton-Dickinson | 381547 | |
Ketamine | Fort Dodge | NDC #0856-2013 | |
Level | Swanson | LLA001 | |
Luer-Lock tip cap | CML Supply | 909-001 | |
Marcaine | Hospira | NDC #: 0409-1560-10 | |
Metzenbaum Scissors | Adler | N/A | |
Micro-introducer (5F) | Galt | KIT 002-01 | |
Mosquito Forceps | Adler | N/A | |
Motor | Herbach and Rademan | H1-08 | |
Oxymorphone | Endo Labs | NDC: 63481-624-10 | |
PKH26 Dye Kit | Sigma | PKH26GL-1KT | |
Porcine Serum | Gemini Bio-Products | 100-115 | 2% concentration in full growth medium |
Potts Scissors | Adler | N/A | |
Precise Vista Skin Stapler | 3M | 3998 | |
PVC Tubing | McMaster-Carr/Insultab | 7132K117 | expanded ID 15.88 mm, recovered ID 7.95 mm |
Right Angle Medium Size | Adler | N/A | |
Scalpel Blade (#10-15) | Bard | 373910 | |
Silicone Tubing | McMaster-Carr | 51735K26 | 16.64 mm OD, 9.52 mm ID |
Syringe (5 cc) | Becton-Dickinson (BD) | 309603 | |
Tegaderm | 3M | 90001 | |
Three-way Stopcock | Kendall | 170060 | |
Ti Tube | Tico Titanium | N/A | Specified as ½” OD, .065″ wall, .370″ ID, .1737 lbs/ft |
Trypsin | Lonza | CC-5012 | |
Trypsin Neutralizing Solution (TNS) | Lonza | CC-5002 | 0.03% |
Vetropolycin | Pharmaderm Animal Health | NAC #: 12920110 | |
Vicryl Suture (3-0) | Ethicon | J808T | |
Water Bath Sonicator | Branson | B200 |