Toshiharu Shin'oka

Transplantation of a Tissue-Engineered Pulmonary Artery

To the Editor: Various vascular grafts are commonly used in the reconstruction of cardiovascular tissues. However, prosthetic or bioprosthetic materials lack growth potential and therefore in child...

Tissue engineering heart valves: Valve leaflet replacement study in a lamb model

BACKGROUND Valve replacements using either bioprosthetic or mechanical valves have the disadvantage that these structures are unable to grow, repair, or remodel and are both thrombogenic and susceptible to infection. These characteristics have significantly limited their durability and longevity. In an attempt to begin to overcome these shortcomings, we have tested the feasibility of constructing heart valve leaflets in lambs by seeding a synthetic polyglycolic acid fiber matrix in vitro with fibroblasts and endothelial cells. METHODS Mixed cell populations of endothelial cells and fibroblasts were isolated from explanted ovine arteries. Endothelial cells were selectively labeled with an acetylated low-density lipoprotein marker and separated from the fibroblasts using a fluorescent activated cell sorter. A synthetic biodegradable scaffold constructed from polyglycolic acid fibers was seeded with fibroblasts, which grew to form a tissue-like sheet. This tissue was subsequently seeded with endothelial cells, which formed a cellular monolayer coating around the leaflet. Using these constructs, autologous (n = 3) and allogenic (n = 4) tissue engineered leaflets were implanted in 7 animals. In each animal the right posterior leaflet of the pulmonary valve was resected and replaced with an engineered valve leaflet. RESULTS All animals survived the procedure. Postoperative echocardiography demonstrated no evidence of stenosis and trivial pulmonary regurgitation in the autografts and moderate regurgitation in the allogenic valves. Collagen analysis of the constructs showed development of an extracellular matrix. Histologic evaluation of the constructs demonstrated appropriate cellular architecture. CONCLUSIONS This preliminary experiment showed that a tissue engineered valve leaflet constructed from its cellular components can function in the pulmonary valve position. Tissue engineering of a heart valve leaflet is feasible, and these preliminary studies suggest that autograft tissue will probably be superior to allogenic tissue.

Successful application of tissue engineered vascular autografts: clinical experience.

Foreign materials often used in cardiovascular surgery may cause unwanted side effects and reduced growth potential. To resolve these problems, we have designed a tissue-engineering technique that utilizes bone marrow cells (BMCs) in clinical treatments. To obtain tissue-engineered material, we harvested saphenous vein samples from patients, which were then minced, cultured and seeded onto a biodegradable scaffold. The first operation was performed in May 1999 as previously described (N. Engl. J. Med. 344 (7) (2001) 532) and this method was repeated on two other patients. From November 2001, we used aspirated BMCs as the cell source, which were seeded onto the scaffold on the day of surgery. This method was applied in 22 patients. There was no morbidity such as thrombogenic complications, stenosis or obstruction of tissue-engineered autografts, and no mortality due to these techniques. These results indicate that BMCs seeded onto a biodegradable scaffold to establish tissue-engineered vascular autografts (TEVAs) is an ideal strategy, and present strong evidence for the justification and validity of our protocol in clinical trials of tissue engineering.

Higher hematocrit improves cerebral outcome after deep hypothermic circulatory arrest

BACKGROUND Various degrees of hemodilution are currently in clinical use during deep hypothermic circulatory arrest to counteract deleterious rheologic effects linked with brain injury by previous reports. MATERIAL AND METHODS Seventeen piglets were randomly assigned to three groups. Group I piglets (n = 7) received colloid and crystalloid prime (hematocrit < 10%), group II piglets (n = 5) received blood and crystalloid prime (hematocrit 20%), group III piglets (n = 5) received blood prime (hematocrit 30%). All groups underwent 60 minutes of deep hypothermic circulatory arrest at 15 degrees C with continuous magnetic resonance spectroscopy and near-infrared spectroscopy Neurologic recovery was evaluated for 4 days (neurologic deficit score 0, normal, to 500, brain death; overall performance category 1, normal, to 5, brain death). Neurohistologic score (0, normal, to 5+, necrosis) was assessed after the animals were euthanized on day 4. RESULTS Group I had significant loss of phosphocreatine and intracellular acidosis during early cooling (phosphocreatine in group I, 86.3% +/- 26.8%; group II, 117.3% +/- 8.6%; group III, 110.9% +/- 2.68%; p = 0.0008; intracellular pH in group I, 6.95 +/- 0.18; group II, 7.28 +/- 0.04; group III, 7.49 +/- 0.04; p = 0.0048). Final recovery was the same for all groups. Cytochrome aa3 was more reduced in group I during deep hypothermic circulatory arrest than in either of the other groups (group I, -43.6 +/- 2.6; group II, -16.0 +/- 5.2; group III, 1.3 +/= 3.1; p < 0.0001). Neurologic deficit score was best preserved in group III (p < 0.05 group II vs group III) on the first postoperative day, although this difference diminished with time and all animals were neurologically normal after 4 days. Histologic assessment was worst among group I in neocortex area (group I, 1.33 +/- 0.3; group II, 0.22 +/- 0.1; group III, 0.40 +/- 0.2, p < 0.05, group I vs group II; p = 0.0287, group I vs group III). CONCLUSION Extreme hemodilution during cardiopulmonary bypass may cause inadequate oxygen delivery during early cooling. The higher hematocrit with a blood prime is associated with improved cerebral recovery after deep hypothermic circulatory arrest.

The in vitro construction of a tissue engineered bioprosthetic heart valve.

PROBLEM Heart valve replacement with either a nonliving xenograft or a mechanical prosthesis is an effective therapy for valvular heart disease. Both of these approaches have limitations, including their inability to grow, repair, and remodel. In addition, a mechanical prosthesis requires long-term anticoagulation therapy. METHODS This study demonstrates the in vitro creation of tissue engineered heart valve tissue using cardiovascular cells on degradable polymer matrices, 40 heart valve leaflets were created using this technique from two sources. Xenograft leaflets were created using human dermal fibroblasts and bovine aortic endothelial cells (n = 20) or allograft valve leaflets were created using sheep myofibroblasts and sheep endothelial cells (n = 20). A mixed sheep cell population was obtained consisting of endothelial cells and myofibroblasts. Endothelial cells were labelled with acethylated low density lipoprotein (Ac-Dil-LDL) and cells were separated into two groups using an activated cell sorter: LDL positive cells comprised of a pure endothelial cell population and LDL negative cells comprised of mixed cell population containing myofibroblasts and smooth muscle cells. The LDL negative cells were seeded on a synthetic polyglycolic acid (PGA) mesh and grown in vitro to form a tissue-like fibroblast-mesh core. Endothelial cells were then seeded onto the surface of the fibroblast-mesh core, forming a single monolayer. RESULTS Histological evaluation of these constructs revealed an inner core of LDL negative cells and outer endothelial-like cells which were factor VIII positive. There was no evidence of capillary formation from endothelial cells invading the myofibroblasts and smooth muscle matrix and the endothelial lining appeared complete. CONCLUSIONS It is feasible to construct allogenic heart valve tissue which could be used to make a valve.

Postischemic hyperthermia exacerbates neurologic injury after deep hypothermic circulatory arrest

BACKGROUND Aggressive surface warming is a common practice in the pediatric intensive care unit. However, recent rodent data emphasize the protective effect of mild (2 degrees - 3 degrees C) hypothermia after cerebral ischemia. This study evaluates different temperature regulation strategies after deep hypothermic circulatory arrest with a survival piglet model. METHODS Fifteen piglets were randomly assigned to 3 groups. All groups underwent 100 minutes of deep hypothermic circulatory arrest at 15 degrees C. Brain temperature was maintained at 34 degrees C for 24 hours after cardiopulmonary bypass in group I, 37 degrees C in group II, and 40 degrees C in group III. Neurobehavioral recovery was evaluated daily for 3 days after extubation by neurologic deficit score (0, normal; 500, brain death) and overall performance category (1, normal; 5, brain death). Histologic examination was assessed for hypoxic-ischemic injury (0, normal; 5, necrosis) in a blinded fashion. RESULTS All results are expressed as mean +/- standard deviation. Recovery of neurologic deficit score (12.0 +/- 17.8, 47.0 +/- 49.95, 191.0 +/- 179.83; P = .05 for group I vs III), overall performance category (1.0 +/- 0.0, 1.4 +/- 0.6, 2.8 +/- 1.3; P < .05 for group I vs III), and histologic scores (0.0 +/- 0.0, 1.0 +/- 1.2, 2.8 +/- 1.8; P < .05 for group I vs III cortex) were significantly worse in hyperthermic group III. These findings were associated with a significantly lower cytochrome aa3 recovery determined by near-infrared spectroscopy in group III animals (P = .0041 for group I vs III). No animal recovered to baseline electroencephalographic value by 48 hours after deep hypothermic circulatory arrest. Recovery was significantly delayed in the hyperthermic group III animals, with a lower amplitude 14 hours after the operation, which gradually increased with time (P < .05 for group III vs groups I and II). CONCLUSIONS Mild postischemic hyperthermia significantly exacerbates functional and structural neurologic injury after deep hypothermic circulatory arrest and should therefore be avoided.

Tissue engineering lamb heart valve leaflets

Tissue engineered lamb heart valve leaflets (N − 3) were constructed by repeatedly seeding a concentrated suspension of autologous myofibroblasts onto a biodegradable synthetic polymeric scaffold composed of fibers made from polyglycolic acid and polylactic acid. Over a 2‐week period the cells attached to the polymer fibers, multiplied, and formed a tissue core in the shape of the matrix. The tissue core was seeded with autologous large‐vessel endothelial cells that formed a monolayer which coated the outer surface of the leaflet. The tissue engineered leaflets were surgically implanted in place of the right posterior pulmonary valve leaflet of the donor lamb while on cardiopulmonary bypass. Pulmonary valve function was evaluated by two‐dimensional echocardiography with color Doppler which demonstrated valve function without evidence of stenosis and with only trivial regurgitation under normal physiologic conditions. Histologically, the tissue engineered heart valve leaflets resembled native valve leaflet tissue.

Tissue-engineered vascular grafts for use in the treatment of congenital heart disease: from the bench to the clinic and back again.

Since the first tissue-engineered vascular graft (TEVG) was implanted in a child over a decade ago, growth in the field of vascular tissue engineering has been driven by clinical demand for improved vascular prostheses with performance and durability similar to an autologous blood vessel. Great strides were made in pediatric congenital heart surgery using the classical tissue engineering paradigm, and cell seeding of scaffolds in vitro remained the cornerstone of neotissue formation. Our second-generation bone marrow cell-seeded TEVG diverged from tissue engineering dogma with a design that induces the recipient to regenerate vascular tissue in situ. New insights suggest that neovessel development is guided by cell signals derived from both seeded cells and host inflammatory cells that infiltrate the graft. The identification of these signals and the regulatory interactions that influence cell migration, phenotype and extracellular matrix deposition during TEVG remodeling are yielding a next-generation TEVG engineered to guide neotissue regeneration without the use of seeded cells. These developments represent steady progress towards our goal of an off-the-shelf tissue-engineered vascular conduit for pediatric congenital heart surgery.

Tissue engineering of cardiovascular structures

Congenital and acquired diseases of the heart valves and great arteries are leading causes of morbidity and mortality. Current prosthetic or bioprosthetic replacement devices are imperfect and subject patients to one or more ongoing risks including thrombosis, limited durability, increased susceptibility to infection, and need for reoperations due to lack of growth. Tissue engineering (TE) is a new discipline that offers the potential to create replacement structures from autologous cells and biodegradable polymers. Because TE constructs contain living cells, they may have the potential for growth and self-repair and remodeling. Cardiac valve leaflets and large conduit arteries have been made with the TE approach. These TE structures have functioned in the pulmonary circulation of growing lambs for up to 4 months and have demonstrated 1) structural organization to resemble normal valve and artery, 2) satisfactory physiologic function, 3) lack of thrombus formation, and 4) growth.

Oxygenation strategy and neurologic damage after deep hypothermic circulatory arrest. II. Hypoxic versus free radical injury

OBJECTIVES Laboratory studies suggest that myocardial reperfusion injury is exacerbated by free radicals when pure oxygen is used during cardiopulmonary bypass. In phase I of this study we demonstrated that normoxic perfusion during cardiopulmonary bypass does not increase the risk of microembolic brain injury so long as a membrane oxygenator with an arterial filter is used. In phase II of this study we studied the hypothesis that normoxic perfusion increases the risk of hypoxic brain injury after deep hypothermia with circulatory arrest. METHODS With membrane oxygenators with arterial filters, 10 piglets (8-10 kg) underwent 120 minutes of deep hypothermia and circulatory arrest at 15 degrees C, were rewarmed to 37 degrees C, and were weaned from bypass. In 5 piglets normoxia (PaO2 64-181 mm Hg) was used during cardiopulmonary bypass and in 5 hyperoxia (PaO2 400-900 mm Hg) was used. After 6 hours of reperfusion the brain was fixed for histologic evaluation. Near-infrared spectroscopy was used to monitor cerebral oxyhemoglobin and oxidized cytochrome a,a3 concentrations. RESULTS Histologic examination revealed a significant increase in brain damage in the normoxia group (score 12.4 versus 8.6, P =.01), especially in the neocortex and hippocampal regions. Cytochrome a,a 3 and oxyhemoglobin concentrations tended to be lower during deep hypothermia and circulatory arrest in the normoxia group (P =.16). CONCLUSIONS In the setting of prolonged deep hypothermia and circulatory arrest with membrane oxygenators, normoxic cardiopulmonary bypass significantly increases histologically graded brain damage with respect to hyperoxic cardiopulmonary bypass. Near-infrared spectroscopy suggests that the mechanism is hypoxic injury, which presumably overwhelms any injury caused by increased oxygen free radicals.