Jason S. Sperling

Tissue-engineered valved conduits in the pulmonary circulation.

OBJECTIVE Bioprosthetic and mechanical valves and valved conduits are unable to grow, repair, or remodel. In an attempt to overcome these shortcomings, we have evaluated the feasibility of creating 3-leaflet, valved, pulmonary conduits from autologous ovine vascular cells and biodegradable polymers with tissue-engineering techniques. METHODS Endothelial cells and vascular medial cells were harvested from ovine carotid arteries. Composite scaffolds of polyglycolic acid and polyhydroxyoctanoates were formed into a conduit, and 3 leaflets (polyhydroxyoctanoates) were sewn into the conduit. These constructs were seeded with autologous medial cells on 4 consecutive days and coated once with autologous endothelial cells. Thirty-one days (+/-3 days) after cell harvesting, 8 seeded and 1 unseeded control constructs were implanted to replace the pulmonary valve and main pulmonary artery on cardiopulmonary bypass. No postoperative anticoagulation was given. Valve function was assessed by means of echocardiography. The constructs were explanted after 1, 2, 4, 6, 8, 12, 16, and 24 weeks and evaluated macroscopically, histologically, and biochemically. RESULTS Postoperative echocardiography of the seeded constructs demonstrated no thrombus formation with mild, nonprogressive, valvular regurgitation up to 24 weeks after implantation. Histologic examination showed organized and viable tissue without thrombus. Biochemical assays revealed increasing cellular and extracellular matrix contents. The unseeded construct developed thrombus formation on all 3 leaflets after 4 weeks. CONCLUSION This experimental study showed that valved conduits constructed from autologous cells and biodegradable matrix can function in the pulmonary circulation. The progressive cellular and extracellular matrix formation indicates that the remodeling of the tissue-engineered structure continues for at least 6 months.

New Pulsatile Bioreactor for In Vitro Formation of Tissue Engineered Heart Valves

Two potential obstacles to the creation of implantable tissue engineered heart valves are inadequate mechanical properties (ability to withstand hemodynamic stresses) and adverse host-tissue reactions due to the presence of residual nondegraded polymer scaffold. In an attempt to address these problems, we developed an in vitro cell culture system that provides physiological pressure and flow of nutrient medium to the developing valve constructs. It is anticipated that in vitro physical stress will stimulate the tissue engineered heart valve construct to develop adequate strength prior to a possible implantation. Long-term in vitro development will be realized by an isolated and thereby contamination-resistant system. Longer in vitro development will potentially enable more complete biodegradation of the polymeric scaffold during in vitro cultivation. This new dynamic bioreactor allows for adjustable pulsatile flow and varying levels of pressure. The system is compact and easily fits into a standard cell incubator, representing a highly isolated dynamic cell culture setting with maximum sterility, optimal gas supply and stable temperature conditions especially suited for long-term experiments.

Tissue engineering of heart valves: in vitro experiences.

BACKGROUND Tissue engineering is a new approach, whereby techniques are being developed to transplant autologous cells onto biodegradable scaffolds to ultimately form new functional tissue in vitro and in vivo. Our laboratory has focused on the tissue engineering of heart valves, and we have fabricated a trileaflet heart valve scaffold from a biodegradable polymer, a polyhydroxyalkanoate. In this experiment we evaluated the suitability of this scaffold material as well as in vitro conditioning to create viable tissue for tissue engineering of a trileaflet heart valve. METHODS We constructed a biodegradable and biocompatible trileaflet heart valve scaffold from a porous polyhydroxyalkanoate (Meatabolix Inc, Cambridge, MA). The scaffold consisted of a cylindrical stent (1 x 15 x 20 mm inner diameter) and leaflets (0.3 mm thick), which were attached to the stent by thermal processing techniques. The porous heart valve scaffold (pore size 100 to 240 microm) was seeded with vascular cells grown and expanded from an ovine carotid artery and placed into a pulsatile flow bioreactor for 1, 4, and 8 days. Analysis of the engineered tissue included biochemical examination, enviromental scanning electron microscopy, and histology. RESULTS It was possible to create a trileaflet heart valve scaffold from polyhydroxyalkanoate, which opened and closed synchronously in a pulsatile flow bioreactor. The cells grew into the pores and formed a confluent layer after incubation and pulsatile flow exposure. The cells were mostly viable and formed connective tissue between the inside and the outside of the porous heart valve scaffold. Additionally, we demonstrated cell proliferation (DNA assay) and the capacity to generate collagen as measured by hydroxyproline assay and movat-stained glycosaminoglycans under in vitro pulsatile flow conditions. CONCLUSIONS Polyhydroxyalkanoates can be used to fabricate a porous, biodegradable heart valve scaffold. The cells appear to be viable and extracellular matrix formation was induced after pulsatile flow exposure.

Technical Report: Fabrication of a Trileaflet Heart Valve Scaffold from a Polyhydroxyalkanoate Biopolyester for Use in Tissue Engineering

Previously, we reported the implantation of a single tissue engineered leaflet in the posterior position of the pulmonary valve in a lamb model. The major problems with this leaflet replacement were the scaffolds inherent stiffness, thickness, and nonpliability. We have now created a scaffold for a trileaflet heart valve using a thermoplastic polyester. In this experiment, we show the suitability of this material in the production of a biodegradable, biocompatible scaffold for tissue engineered heart valves. A heart valve scaffold was constructed from a thermoplastic elastomer. The elastomer belongs to a class of biodegradable, biocompatible polyesters known as polyhydroxyalkanoates (PHAs) and is produced by fermentation (Metabolix Inc., Cambridge, MA). It was modified by a salt leaching technique to create a porous, three-dimensional structure, suitable for tissue engineering. The trileaflet heart valve scaffold consisted of a cylindrical stent (1 mm X 15 mm X 20 mm I.D.) containing three valve leaflets. The leaflets were formed from a single piece of PHA (0.3 mm thick), and were attached to the outside of the stent by thermal processing techniques, which required no suturing. After fabrication, the heart valve construct was allowed to crystallize (4 degrees C for 24 h), and salt particles were leached into doubly distilled water over a period of 5 days to yield pore sizes ranging from 80 to 200 microns. Ten heart valve scaffolds were fabricated and seeded with vascular cells from an ovine carotid artery. After 4 days of incubation, the constructs were examined by scanning electron microscopy. The heart valve scaffold was tested in a pulsatile flow bioreactor and it was noted that the leaflets opened and closed. Cells attached to the polymer and formed a confluent layer after incubation. One advantage of this material is the ability to mold a complete trileaflet heart valve scaffold without the need for suturing leaflets to the conduit. Second advantage is the use of only one polymer material (PHA) as opposed to hybridized polymer scaffolds. Furthermore, the mechanical properties of PHA, such as elasticity and mechanical strength, exceed those of the previously utilized material. This experiment shows that PHAs can be used to fabricate a three-dimensional, biodegradable heart valve scaffold.

Evaluation of biodegradable, three-dimensional matrices for tissue engineering of heart valves.

A crucial factor in tissue engineering of heart valves is the type of scaffold material. In the following study, we tested three different biodegradable scaffold materials, polyglycolic acid (PGA), polyhydroxyalkanoate (PHA), and poly-4-hydroxybutyrate (P4HB), as scaffolds for tissue engineering of heart valves. We modified PHA and P4HB by a salt leaching technique to create a porous matrix. We constructed trileaflet heart valve scaffolds from each polymer and tested them in a pulsatile flow bioreactor. In addition, we evaluated the cell attachment to our polymers by creating four tubes of each material (length equals 4 cm; inner diameter, 0.5 cm), seeding each sample with 8,000,000 ovine vascular cells, and incubating the cell-polymer construct for 8 days (37 degrees C and 5% CO2). The seeded vascular constructs were exposed to continuous flow for 1 hour. Analysis of samples included DNA assay before and after flow exposure, 4-hydroxyproline assay, and environmental scanning electron microscopy (ESEM). We fabricated trileaflet heart valve scaffolds from porous PHA and porous P4HB, which opened and closed synchronously in a pulsatile bioreactor. It was not possible to create a functional trileaflet heart valve scaffold from PGA. After seeding and incubating the PGA-, PHA-, and P4HB-tubes, there were significantly (p < 0.001) more cells on PGA compared with PHA and P4HB. There were no significant differences among the materials after flow exposure, but there was a significantly higher collagen content (p < 0.017) on the PGA samples compared with P4HB and PHA. Cell attachment and collagen content was significantly higher on PGA samples compared with PHA and P4HB. However, PHA and P4HB also demonstrate a considerable amount of cell attachment and collagen development and share the major advantage that both materials are thermoplastic, making it possible to mold them into the shape of a functional scaffold for tissue engineering of heart valves.

Stroke-Related Mortality in Coronary Surgery Is Reduced by the Off-Pump Approach

BACKGROUND Stroke remains an important complication after coronary artery bypass graft surgery (CABG). We sought to determine the frequency and death-related incidence of stroke after on-pump and off-pump CABG. METHODS We analyzed 4,869 consecutive isolated CABG performed in our institution. Of these, 3,490 (71.7%) were off-pump and 1,379 (28.3%) were on-pump. Propensity matched samples of 1,379 off-pump and 1,379 on-pump were compared on clinical presentation and The Society of Thoracic Surgeons (STS) predicted scores for risk of postoperative mortality and stroke. Univariate analyses were used to compare the relationship of off-pump and on-pump groups to postoperative mortality and stroke. Multivariate logistic regression was used to determine the unique association between all variables and occurrence of mortality after stroke. RESULTS No differences were found for sex, diabetes mellitus, history of renal failure, prior stroke, or timing of surgery. Postoperative mortality occurred in 75 patients (2.7%) and stroke in 47 (1.7%). The off-pump patients had a lower rate of stroke (1.0% versus 2.4%; p < 0.01) compared with on-pump patients. Mortality after stroke occurred in 14 patients, with a lower rate occurring in the off-pump group (14.3% versus 36.4%; p = 0.07). Multivariate analyses controlling for the effect of preoperative risk factors and STS mortality risk demonstrated that off-pump status was independently associated with an 84% decrease in the risk of death after stroke (adjusted odds ratio 0.157, 95% confidence interval: 0.035 to 0.711, p = 0.016). CONCLUSIONS Off-pump CABG is associated with lower stroke rates and stroke-related mortality. It may be useful to consider off-pump CABG for patients who are at higher risk for postoperative stroke.

Impact of New York Heart Association classification, advanced age and patient-prosthesis mismatch on outcomes in aortic valve replacement surgery

OBJECTIVES More elderly patients (>80 years of age) are being referred for aortic valve replacement (AVR) with or without CABG. Current risk stratification models may not accurately predict the preoperative risk in these patients. We sought to determine which perioperative variables were relevant in determining short-term (30-day to in-hospital) outcomes in our intuitions series of consecutive AVR and AVR+CABG surgeries. We constructed a novel variable, patient-prosthesis mismatch (PPM) in the presence of diminished functional status (NYHA) classification, and studied its role as a predictor of mortality risk. METHODS From 2006 to 2010, 509 patients undergoing AVR or AVR+CABG were evaluated. We created four groups based on the age and procedure (AVR >80, AVR+CABG >80, AVR <80 and AVR+CABG <80). PPM was defined as a calculated effective orifice area index value of ≤ 0.85, and it was calculated from manufacturer-generated charts. In-hospital and 30-day outcomes were assessed using the Chi-square and logistic regression analyses. RESULTS Overall observed 30-day mortality for all groups was lower (n = 8, 1.6%) than the STS-predicted mortality. Reoperation and PPM+NYHA class III-IV were associated with short-term mortality, but age >80 years was not. Octogenarians referred for surgery often had advanced heart failure. CONCLUSIONS Overall, short-term outcomes after AVR with or without CABG were excellent and lower than predicted by the STS model. The low risk of AVR with CABG supports the consideration for earlier surgical referral and intervention for patients with a high likelihood of aortic stenosis progression before the onset of advanced heart failure ensues, regardless of the age. This should help further decrease the already very low mortality observed in these series. Efforts to avoid PPM in the setting of advanced heart failure may improve short-term results in this subset of patients.

Effects of cyanosis and hypothermic circulatory arrest on lung function in neonatal lambs

BACKGROUND Lung function is often impaired after cardiac surgery and cardiopulmonary bypass (CPB), particularly in chronically cyanotic patients. This study aimed to evaluate lung function in a surgically created chronic cyanotic neonatal lamb model after CPB and deep hypothermic circulatory arrest (DHCA) and to assess the role of nitric oxide (NO) in the pathogenesis of increased pulmonary vascular resistance. METHODS A chronic cyanosis model was surgically created in 7 lambs (4.7+/-0.8 days old) by anastomosing the pulmonary artery (PA) to the left atrium (LA). Another 7 lambs underwent a sham operation (control). One week later, the animals underwent shunt takedown and CPB with 90 minutes of DHCA at 18 degrees C. Cardiac index (CI), pulmonary vascular resistance index (PVRI), lung dynamic compliance (Cdyn), alveolar-arterial oxygen difference (AaDO2), left atrial plasma nitrate/nitrite (NO metabolites) levels, and pulmonary cGMP production (concentration difference between LA and PA) were measured before CPB and at 1 and 2 hours after reperfusion. RESULTS The cyanosis model consistently produced significantly lower arterial oxygen tension (34.8+/-2.3 vs 93.1+/-8.8 torr in control, p < 0.001) and Qp/Qs (0.6+/-0.1 vs 1.0+/-0.0 in control, p < 0.001) than controls. Postoperative PVRI was significantly lower in the cyanosis group than in controls, although CPB with DHCA significantly elevated PVR in both cyanotic and control animals. There were no significant differences in AaDO2 and Cdyn after CPB between groups. The level of NO metabolites did not change before or after CPB in either cyanotic or acyanotic animals. NO metabolite levels tended to be higher in the cyanotic animals (p = 0.08). There was no significant difference in pulmonary cGMP production between both groups. CONCLUSIONS These findings suggest that CPB with DHCA, per se, does not affect NO production in cyanotic or acyanotic neonatal lambs but causes increased PVR in both groups. Chronic cyanosis does not result in reduced pulmonary function after CPB with DHCA, and is associated with lower PVR. The mechanism may involve an increased NO production in cyanotic animals.