Andreina Schoeberlein

Tissue engineering in cardiovascular surgery : MTT, a rapid and reliable quantitative method to assess the optimal human cell seeding on polymeric meshes

OBJECTIVE Currently used valve substitutes for valve replacement have certain disadvantages that limit their long-term benefits such as poor durability, risks of infection, thromboembolism or rejection. A tissue engineered autologous valve composed of living tissue is expected to overcome these shortcomings with natural existing biological mechanisms for growth, repair, remodeling and development. The aim of the study was to improve cell seeding methods for developing tissue-engineered valve tissue. METHODS Human aortic myofibroblasts were seeded on polyglycolic acid (PGA) meshes. Cell attachment and growth of myofibroblasts on the PGA scaffolds with different seeding intervals were compared to determine an optimal seeding interval. In addition, scanning electron microscopy study of the seeded meshes was also performed to document tissue development. RESULTS There was a direct correlation between cell numbers assessed by direct counting and MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltertra-zoliu m bromide) assay. Both attach rate and cell growth seeded on meshes with long intervals (24 and 36 h) were significantly higher than those seeded with short intervals (2 and 12 h) (P<0.01), there was no significant difference between 24- and 36-h seeding interval. Scanning electron microscopy also documented more cell attachment with long seeding intervals resulting in a more solid tissue like structure. CONCLUSION It is feasible to use human aortic myofibroblasts to develop a new functional tissue in vitro. Twenty-four hours is an optimal seeding interval for seeding human aortic myofibroblasts on PGA scaffolds and MTT test is a rapid and reliable quantitative method to assess the optimal human cell seeding on polymeric meshes.

Tissue engineering: A new approach in cardiovascular surgery; Seeding of human fibroblasts followed by human endothelial cells on resorbable mesh

OBJECTIVE In tissue engineering the material properties of synthetic compounds are chosen to enable delivery of dissociated cells onto a scaffold in a manner that will result in in vitro formation of a new functional tissue. The seeding of human fibroblasts followed by human endothelial cells on resorbable mesh is a precondition of a successful creation of human tissues such as vessels or cardiac valves. METHODS Polymeric scaffolds (n = 18) composed of polyglycolic acid (PGA) with a fiber diameter of 12-15 microm and a polymer density of 70 mg/ml were used as square sheets of 1 x 1 x 0.3 cm. Fibroblasts (passage 7) harvested from human foreskin were seeded (3.4 x 10(6)) and cultured over a 3 week period on a PGA-mesh, followed by seeding of endothelial cells (passage 5, 2.8 x 10(6)) harvested from human ascending aorta. Thereafter the new tissue was stained for HE, van Gieson, Trichrom Masson, Factor VIII and CD 34 and proved by scanning electron microscopy. RESULTS Microscopic examination of the seeded mesh demonstrated that the human fibroblasts were attached to the polymeric fibers and had begun to spread out and divide. The scanning electron microscopic examination demonstrated a homogeneous scaffold resembling a solid sheet of tissue. The seeded endothelial cells formed a monolayer on the fibroblasts and no endothelial cell invasion or new formation of capillaris could be detected. CONCLUSIONS These results are a first step to demonstrate that seeding of human fibroblasts and endothelial cells on PGA-mesh might be a feasible model to construct human tissues such as vessels or cardiac valves.

Modified ultrafiltration lowers adhesion molecule and cytokine levels after cardiopulmonary bypass without clinical relevance in adults.

OBJECTIVE Cardiac surgery with cardiopulmonary bypass (CPB) results in expression of cytokines and adhesion molecules (AM) with subsequent inflammatory response. The purpose of the study was to evaluate the clinical impact of modified ultrafiltration (MUF) and its efficacy in reducing cytokines and AM following coronary artery bypass grafting (CABG) in adults. METHODS A prospective randomized study of 97 patients undergoing elective CABG was designed. Fifty patients were operated on using normothermic and 47 patients using hypothermic CPB. The normothermic group was subdivided into a group with modified ultrafiltration (n = 30) and a group without MUF (n = 20). In the hypothermic group 30 patients received MUF compared to 17 patients serving as controls. MUF was instituted after CPB for 15 min through the arterial and venous bypass circuit lines. Cytokines (IL-6, IL-8, TNF-alpha, IL-2R) and adhesion molecules (sE-selectin, sICAM-1) were measured preoperatively, pre-MUF, in the ultrafiltrate, 24 h, 48 h and 6 days after surgery by chemiluminescent enzyme immunometric assay or enzyme-linked immunosorbent assay (ELISA). Clinical parameters were collected prospectively until discharge. RESULTS In all patients AM and cytokines were significantly elevated after normothermic and hypothemic CPB. AM and cytokines were significantly higher in hypothermia compared to normothermia. In hypothermic CPB sE-selectin was decreased after 24 h by 37% (P < 0.0063) and by 40% (P < 0.0027) after 48 h postoperatively. ICAM-1 was reduced by 43% (P < 0.0001) after 24 h and by 60% (P < 0.0001) after 6 days. Similar results were seen in cytokines with reduction up to 60% after 24 h. Changes after 48 h were noticeable but not significant. Reduction of AM and cytokines after normothermic CPB was minimal. Neither in normothermia, nor in hypothermia has sIL-2R been effectively removed from the circulation. There were no significant differences in the clinical variables between the patients with or without MUF. CONCLUSION AM and cytokines are significantly elevated after hypothermic CPB compared to normothermic CPB. MUF led to a significant reduction in cytokine and AM levels after hypothermic CPB, except for IL-2R. MUF showed minimal effect in normothermia. We conclude that MUF is an efficient way to remove cytokines and AM. However, we were unable to demonstrate any significant impact of MUF in outcome of adults after elective CABG.

Expression of adhesion molecules and cytokines after coronary artery bypass grafting during normothermic and hypothermic cardiac arrest

OBJECTIVE Cardiac surgery with cardiopulmonary bypass (CPB) results in vascular injury and tissue damage which involves leukocyte-endothelial interactions mediated by cytokines and adhesion molecules. This study was designed to demonstrate the effect of normothermic and hypothermic CPB to cytokine and soluble adhesion molecule levels in adults and to determine whether these levels correlate to the patients postoperative course. DESIGN AND PATIENTS In 25 patients after normothermic and in 25 patients after hypothermic coronary artery bypass grafting with cardiopulmonary bypass (CPB), blood samples for cytokine and soluble adhesion molecule analysis were taken preoperatively, 24, 36, 48 h, and 6 days postoperatively. Soluble adhesion molecules (sE-selectin, sICAM-1) were measured by ELISA and cytokines (TNF-alpha, IL-6, IL-8) by chemilumenscent-immunoassay. Clinical data were collected prospectively. RESULTS Postoperatively, adhesion molecule and cytokine levels were significantly elevated after CPB. Mean plasma levels of sICAM-1 was 2.4-fold higher after 6 days. Mean plasma concentration of sE-selectin peaked after 48 h with a 2-fold increase compared to normothermic conditions. In the hypothermia group sICAM-1, sE-selectin, IL-6, and IL-8 showed significantly higher levels (P<0.0057, P<0.0012, P<0.0419, P<0.0145) after 24 h compared to the normothermia group. No clinical differences were seen. CONCLUSION Adhesion molecules and cytokines are elevated after CPB. Patients after hypothermic CPB show significant higher sICAM-1, sE-selectin, IL-6, and IL-8 levels after 24 h compared to normothermic conditions. These results are mainly due to longer CPB and crossclamp times but do not alter the patients postoperative course.

Scaffold Precoating with Human Autologous Extracellular Matrix for Improved Cell Attachment in Cardiovascular Tissue Engineering

Cell attachment to a scaffold is a precondition for the development of bioengineered valves and vascular substitutes. This attachment is generally facilitated by the use of precoating factors, but some can cause toxic or immunologic side effects. Autologous extracellular matrix (ECM) is used as a precoating factor in our study. Ascending aortic tissue was cultured to obtain human myofibroblasts. Autologous ECM was extracted from the same aortic tissue. Poly(glycolic acid) (PGA) scaffolds were precoated with autologous ECM, human serum, or poly-L-lysine; the control group was pretreated with phosphate buffered saline (PBS). Myofibroblasts were seeded onto each scaffold, and the cell attachment was assayed and compared. Compared with the control group, precoating with human serum, poly-L-lysine, and ECM increased number of attached cells by 24%, 53%, and 48%, respectively. Differences between precoating groups were significant (p < 0.01), except for ECM versus poly-L-lysine. Scanning electron microscopy also demonstrated the high degree of cell attachment to the PGA fibers on scaffolds precoated with ECM and poly-L-lysine. Precoating polymeric scaffold with autologous human extracellular matrix is a very effective method of improving cell attachment in cardiovascular tissue engineering without the potential risk of immunologic reactions.

Fluorescence Activated Cell Sorting: A Reliable Method in Tissue Engineering of a Bioprosthetic Heart Valve

BACKGROUND Techniques of tissue engineering are used to seed human autologous cells in vitro on degradable mesh to create new functional tissue like a bioprosthetic heart valve. A precondition is subsequent seeding of native-valve-analogous pure endothelial and myofibroblast cell lines. The aim of this study is to find a safe method of isolating viable cell lines out of tissues from the operating room. METHODS Mixed cells from ascending aorta obtained from the operating room were incubated with an endothelial-specific fluorescent marker. The labeled cells were activated and sorted by flow cytometry. Isolated cell lines were cultured and thereafter square sheets of polymeric scaffold were seeded with myofibroblasts, followed by endothelial cells. The created tissue was stained with hematoxylin and eosin, van Gieson stain, and stains for factor VIII and CD34. RESULTS Control culture samples (n = 25) revealed vital uncontaminated endothelial and myofibroblast cell lines. Microscopy of the seeded meshes (n = 16) demonstrated a tissue-like structure. Van Gieson stain showed production of collagen. Endothelial cells formed a superficial monolayer, demonstrated by factor VIII and CD34; no invasive formation of capillaries was detectable. CONCLUSIONS These results demonstrate that fluorescence activated cell sorting is a reliable and safe method to gain pure vital autologous cell lines out of human mixed cells for subsequent seeding on degradable mesh and that those cells are active to form new tissue.

Tissue engineering of a bioprosthetic heart valve: stimulation of extracellular matrix assessed by hydroxyproline assay.

Creation of an autologous heart valve by tissue engineering offers a promising approach to cardiac surgery. Although we have demonstrated successful formation of native valve analogous tissue in vitro, hemodynamic competence remains a serious problem. The aim of this study was to optimize in vitro formation of collagen as a precondition for mechanical stability of new tissue. Human myofibroblasts were seeded on square sheets of biodegradable scaffolds (control). To stimulate collagen production, one series was cultured with L-ascorbic acid 2-phosphate. In a second series, the seeded scaffolds were subjected to tension by mounting them on a frame. After 4 weeks of culture time, the collagen content of the different series was assessed by hydroxyproline assay. Light and scanning electron microscopy were performed. Hydroxyproline content of the framed scaffolds was 10 times higher than that of the control group (p < 0.05) and 6 times higher than in the unframed scaffolds grown with ascorbic acid (p < 0.05), respectively. Scanning electron microscopy proved extensive formation of solid tissue in the framed samples. These results demonstrate that supplementation of myofibroblast cultures with ascorbic acid, especially if grown on strained scaffolds, significantly increases collagen content, which is crucial for mechanical stability. This concept is a further step toward the creation of a hemodynamically competent autologous heart valve.

Optimized growth conditions for tissue engineering of human cardiovascular structures.

Optimized in vitro formation of strong tissue is a prerequisite for tissue engineering of cardiovascular structures, such as heart valves and blood vessels. This study evaluates different growth media additives as to cell proliferation, extracellular matrix formation, and mechanical characteristics. Biodegradable polymers were seeded with human vascular myofibroblasts. Group A was cultured with standard medium, groups B, C, and D were in addition supplemented with ascorbate, fibroblast growth factor (bFGF), and both respectively. Analysis included histology, electron microsocopy, mechanical testing, and biochemical assays for cell proliferation (DNA) and extracellular matrix (collagen). DNA content increased in all groups, showing significantly more cells in group C and D after 14d. Collagen increased in all groups, except for C. Morphology showed viable, layered cellular tissue, with collagen fibrils after 2w, most pronounced in B and D. Mechanical properties decreased initially, stabilizing after 2w. In conclusion, standard nutrient media were efficient for seeded human vascular cells cultured on biodegradable meshes. Supplementation with bFGF+ascorbate resulted in enhanced early cell proliferation and structurally more mature tissue formation.

Heat Shock Protein Upregulation Lowers Cytokine Levels after Ischemia and Reperfusion

Objective: Studies showed that the expression of heat shock protein 70 (HSP70) by whole-body hyperthermia or warming of the heart is associated with protection against ischemia/reperfusion injury. The aim of this study is to determine a time-related response of HSP70 expression through topical cardiac warming with correlation to cytokine production. Methods: 30 rats were divided into three groups: no heat shock, heat shocked, and controls. Heat shock was performed with 42°C saline solution applied to the heart for 5, 30, and 60 min. HSP70 and cytokines were measured. Results: Heat shock treated animals showed a 1.2-fold increase after 5 min (NS) in HSP70 expression, a 2.0-fold increase (p < 0.02) after 30 min, and a 2.3-fold increase (p < 0.012) after 60 min compared to controls. The IL-1β levels decreased from 14.3 pg/ml (normal controls) to 7.1 pg/ml after 5 min, to 1.6 pg/ml after 30 min (p < 0.002), and to 1.4 pg/ ml after 60 min of heat shock treatment (p < 0.002). The TNF-α levels also decreased, but not significantly. Conclusions: Upregulation of HSP70 through this novel method is instant and detectable within hours. The amount of HSP70 expression induced is time dependent, showing an indirect correlation with cytokine levels. These results suggest that the protective effect of HSP70 is immediate and might be explained by reduced cytokine levels. No prior recovery period is needed.

Reduction of Soluble E-Selectin after Combined Treatment with Simvastatin and Vitamins C and E

Objective: We investigated the influence of treatment with simvastatin and vitamins C and E on serum levels of soluble cell adhesion molecules (sCAM) as markers of atherosclerosis and endothelial activation. Methods: In 11 hypercholesterolemic patients, serum levels of sCAM were measured by enzyme immunoassay before and after 6 weeks of treatment with either simvastatin (20 mg) or vitamin C (1 g) plus vitamin E (600 mg) and after an additional 6 weeks of combined treatment. In 8 of the patients angiography was performed at study entry. Results: Baseline levels of soluble intercellular adhesion molecule-1 (sICAM-1) showed an age-dependent increase (coefficient of correlation 0.71, p = 0.014) and were higher in patients with established atherosclerotic lesions than in patients with no such lesions (369.5 ± 61.8 vs. 238.4 ± 56.4 ng/ml, p = 0.023). Whereas either treatment alone had no influence on the serum levels of sCAM, combined treatment with simvastatin and vitamins C and E reduced serum levels of soluble E-selectin from 43.2 ± 18.9 ng/ml at baseline to 39.7 ± 16.5 ng/ml (p = 0.027) at study end. Although not significant, levels of soluble P-selectin were also reduced from a mean of 125.9 ± 64.1 ng/ml at baseline to 115 ± 64.9 ng/ml (p = 0.1). Serum levels of sICAM-1 and soluble vascular cell adhesion molecule-1 (sVCAM-1) remained unchanged by this treatment. Conclusion: Combined treatment with simvastatin and vitamins C and E reduced serum levels of soluble E-selectin but had no influence on sICAM-1 and sVCAM-1 levels.