Robert T. Tranquillo
The landmark Science publication by Weinberg and Bell in 1986 [1] described an attempt to create a blood vessel by culturing vascular cells in a tube of reconstituted type I collagen gel, igniting the field of vascular tissue engineering which was reinforced by the term “tissue engineering” coined at a National Science Foundation meeting in 1987—the year I started my faculty position at the University of Minnesota.
Human life is sustained by physiological flow of blood in arteriovenous circuits, and when blood flow is significantly attenuated or transiently interrupted, normal biological processes are negatively affected. Synthetic polymers like Dacron have succeeded as arterial grafts for large-diameter vessels, such as the abdominal aorta. However, small-diameter (<6 mm diameter) arterial grafts, primarily as “bypass vascular grafts” to allow blood to bypass narrowed or occluded regions of coronary and peripheral arteries, account for the vast majority of the global clinical demand. Synthetic polymers have generally proved inadequate as bypass grafts, largely due to acute thrombogenicity, anastomotic intimal hyperplasia, aneurysm formation, infection, and progression of atherosclerotic disease associated in poorly understood ways with lower volumetric blood flow rates through smaller arteries. According to 2012 Healthcare Cost and Utilization Project data, 434,000 coronary artery bypass grafts were placed in 190,000 patients in the USA, and 48,000 patients received bypass grafts for peripheral artery disease the same year. Necessarily, cardiovascular surgeons use autologous vessels as bypass grafts with resultant harvest-site morbidity and poor long-term performance in the case of the most frequently used saphenous vein—if the patient still has one after a prior bypass surgery. Many patients with systemic atherosclerosis do not have any autologous vessel suitable for bypass use.
This unmet need combined with the elegance of using cell traction forces to allow entrapped cells to compact a tube of soft collagen gel into a bypass graft as suggested by Weinberg and Bell captured my imagination back in 1987. This keen interest was amplified by the convergence of four scientific fields: rheology, mechanics, transport, and cell behavior. It also provided a pathway to avoid the limitations of autologous graft availability and artificial polymer materials: a product solution based on a completely biological approach. In fact, I had performed pilot experiments during my postdoc aimed at simulating wound contraction in vitro by culturing skin cells (fibroblasts) in a collagen gel. At this early stage, I did not realize the depth of my naivety in attempting to create a “tissue-engineered artery.” However, 37 years later and after several major advances, our research and development programs have led to the formation of a company, Vascudyne Inc., that has achieved the stage of clinical trials using a closely related version of our refinement of the Weinberg and Bell approach. This includes some key advances along the way.
- Minimizing gel adhesion to the mandrel during its cell induced compaction to achieve the circumferential alignment characteristic of native arteries that confers burst strength (inspired by an observation reported by Dr. Nicholas L’Heureux, co-founder of the first vascular tissue engineering company, Cytograft, in his thesis research).
- Switching from collagen gel to fibrin gel—the primary polymer of a blood clot—and from vascular cells to fibroblasts—the collagen-producing cells in wound healing—to harness the wound response of gel-entrapped cells, converting the worthlessly weak tube resulting from a collagen gel to a supra-physiologically strong tube of tissue resulting from a fibrin gel (inspired by my postdoctoral research in modeling wound contraction).
- Decellularizing the tissue tube made from donor fibroblasts into a storable non-immunogenic allograft, that is, a commercializable process and product (a recognition first reported by Dr. Laura Niklasson, co-founder of the other most advanced vascular tissue engineering company, Humacyte, which seeds vascular cells on a synthetic degradable polymer scaffold to achieve a vascular graft). The decellularization step is only sensible if the resulting tube of cell-produced extracellular matrix—predominantly circumferentially aligned collagens—retains its strength, and, crucially, becomes repopulated with the recipient’s cells to become a living vessel in situ. This is indeed the case based on numerous studies performed in large animals and now also in humans.
The resulting “biologically engineered” tube is thus a marriage of classical tissue engineering championed by Langer and Vacanti, where a living tissue replacement is grown in vitro from the patient’s cells, and avant-garde regenerative medicine, where a transplanted material (as in this case) or transplanted cells leads to a functional tissue or organ owing to natural regenerative processes induced by the transplant.
One of the most exciting outcomes of this line of research is the prospect of a cure for congenital heart defects. There are several categories of defects where neonates and infants lack a critical cardiac artery. Presently, all vascular grafts used, including cadaver grafts (a.k.a. homografts), have zero growth capacity. These children face the prospect of multiple open-heart surgeries (and, often, intervening interventions) to up-size the graft as they grow into adulthood, with obvious trauma for the patient and family, and enormous cost to the health care system. We have shown in two published studies that our biologically engineered tube implanted as a pulmonary artery interpositional graft grows somatically with a young lamb into an adult sheep over one year, that is, increased in dimensions—diameter and length—and in total collagen content. This resulted from site-appropriate recellularization (interstitial tissue cells and an endothelium) without macroscopic calcification typical of most biomaterials in a growth model, or a systemic immune response.
As exciting as that prospect is, isolated arterial malformations are very rare. Many more babies, thousand per year in the U.S., are born with a malformed right ventricular outflow tract (RVOT), comprised of the main pulmonary artery and the tri-leaflet pulmonary valve. RVOT malformation is a common feature of tetralogy of Fallot. As for patients with isolated arterial malformations, these patients must endure multiple upsizing of implanted heart valves. Naturally, there is huge motivation to develop a growing heart valve using the biologically engineered tubes that have demonstrated somatic growth potential in lambs. However, there is an order of magnitude increase in the design and function complexity of a heart valve, which acts as a one-way valve to prevent regurgitation of blood into the chamber following ejection, compared to a vascular graft. The first question was how to create a tri-leaflet heart valve and then, from it, a valved conduit for RVOT repair. In Edisonian fashion, started during my first sabbatical in 1999 when I learned about the nascent field of heart valve tissue engineering, we eventually conceived a novel “tri-tube valve” design and demonstrated somatic growth with sustained valve function for one year, although the growth of the leaflets is not yet fully proven. Very recently, we have also demonstrated the same for a valved conduit for RVOT repair, formed by suturing the tri-tube valve within a fourth tube.
The design and function complexity can be addressed by computer-aided design, using finite element analysis to screen valve geometries for adequate coaptation (the valve seal formed from contacting leaflets) under a diastolic pressure gradient and fluid-structure interaction simulation to evaluate the hydrodynamic behavior of a valve design during pulsatile flows with physiological pressure gradient and flow rate waveforms. Thus, classical engineering analysis using state-of-art numerical methods and large-scale computation, along with constitutive strain energy law modeling of the biaxial mechanical behavior of the tube, are vital aspects of a successful biologically engineered RVOT.
Heart valve tissue engineering—the second leg of cardiovascular tissue engineering (vascular, valve, cardiac)—can also impact the adult prosthetic valve options, which are implanted on a similar scale as bypass grafts in adults to obviate the downward spiral to heart failure because of a regurgitant or stenotic heart valve. The current options serve patients well but are not without deficiency: bioprosthetic valves (animal tissue derived) do not have lifelong durability, and mechanical valves require lifelong anticoagulation. The valved conduit previously described for children when up-sized could be the choice for young adults who require a pulmonary valve replacement (e.g., Ross procedure patients), one that confers the potential of lifelong durability without the need for lifelong anticoagulation.
The third leg—cardiac tissue engineering—can also benefit from cell (cardiomyocyte) alignment, as we showed, but entails two more levels of design and function complexity to achieve the holy grail of a “heart patch” to repair a myocardial infarct: a functional microcirculation and electromechanical coupling to the heart. Moreover, unlike decellularized tissue-engineered vascular grafts and valves that can confer function prior to recellularization, a functional heart patch at implantation will require being sufficiently populated by cardiomyocytes, patient-derived cells so as not to induce immune rejection. Maturation of cardiomyocytes derived from induced pluripotent stem cells (a.k.a. “reprogrammed cells” via transfection for selected transcription factors) to exhibit contractile stress comparable to adult cardiomyocytes remains an intensive area of research. All this will be left to the next generation(s) to advance to the point of clinical trial.
Cardiovascular tissue engineering is now demonstrating clinical impact for small-diameter vascular grafts, with the prospect of near-term impact for heart valve replacements (especially pediatric indications), and long-term impact for cardiac tissue restoration. What is next? Predicting, if not controlling, the regeneration that occurs in any cardiovascular implant, and its relationship to the immune response of a specific patient, is a major challenge that will also require substantial advances over the present state. In the future it is likely that massive biomarker mining and artificial intelligence will be needed to meet this challenge, obviating the necessity for a complete understanding of the remarkable dynamical system that is the human body.
References
- C. B. Weinberg and E. Bell, “A blood vessel model constructed from collagen and cultured vascular cells,” Science, vol. 231, no. 4736, pp. 397–400, Jan. 1986, doi: 10.1126/science.2934816.