In vitro study of percutaneous aortic valve replacement: selection of a tissue valve.

BACKGROUND: Selection of the best tissue valve is an essential step before percutaneous aortic valve replacement (PAVR) becomes a clinical reality. The aim of this study was to evaluate in vitro three different tissue valves mounted within the same endovascular stent. METHODS: Thirty stented valves (10 aortic porcine, 10 pulmonary porcine, and 10 pericardial tubular) were sutured within a 32-mm long by 23-mm diameter cobalt-nickel stent. The porcine valves were trimmed down close to the cusps. All valves were delivered with a percutaneous valvuloplasty catheter and placed orthotopically in a latex root that was cast from a sheep's aorta. The roots were tested in a pulse duplicator at a rate of 60 beats per minute and 3.5 liters per minute. The transvalvular gradient, maximum valve orifice area, and presence of central and paravalvular leaks were recorded echocardiographically. RESULTS: Within the limitations of implantation in a synthetic, noncalcified annulus, the pericardial valve performed best in terms of orifice area, transvalvular gradients, and tissue bulk; but four of the ten valves showed a central leak due to the type of stent used. CONCLUSION: The ideal valve for PAVR should collapse with minimal bulk to avoid coronary obstruction and central and paravalvular leaks. The tubular pericardial valve showed the lowest pressure gradients and was the most compressible, but was more open to manufacturing errors.

Mitral valve basal chordae: comparative anatomy and terminology.

BACKGROUND: Recent awareness of the importance of the mitral valve's basal chordae stimulated a comparative anatomic study of these chordae in 11 human, 10 ovine, and 10 porcine hearts. METHODS: The basal chordae were defined as the chordae that arise from the papillary muscles and insert into the ventricular aspect of the leaflets. RESULTS: All leaflet insertions of the basal chordae were close to the annulus, except at the anterior mitral leaflet, where insertion was at the junction of the smooth and rough zones. The number of basal chordae was 24.6 +/- 4.21 in the porcine, 19.7 +/- 2.90 in ovine, and 18.81 +/- 3.54 in the human hearts. At least two anterior basal chordae were present in each half of the anterior leaflet in 70% of ovine and porcine and in 100% of human hearts. At least two basal chordae were present in each half of the middle scallop of the posterior mitral leaflet in 80% of ovine, 70% of porcine, and 63.6% of humans. Among them, only the two principal or strut chordae were identified as the longest and thickest. CONCLUSIONS: The basal chordae of the mitral valve follow a definite pattern in each of the three species studied. A new and logical terminology that should facilitate identification of specific basal chordae is suggested.

Coronary flow obstruction in percutaneous aortic valve replacement. An in vitro study.

OBJECTIVE: Coronary flow obstruction is a serious complication reported in percutaneous aortic valve replacement. In an in vitro study of porcine hearts, the effects of valved stent implantation on coronary artery flow were studied with the native valve's leaflets intact and excised. METHODS: The right and left main coronary arteries of porcine hearts were dissected 20mm distal to the aortic root and directed into lengths of latex tubing leading to collection flasks. The ascending aorta was cut proximal to the brachiocephalic trunk, cannulated, and attached to a constant-head water supply. After steady flow was achieved, the flow rate from each coronary artery was measured. In Group A (n=10), a tubular pericardial valve sutured into a cylindrical, cobalt-nickel stent was deployed orthotopically using a valvuloplasty balloon catheter. In Group B (n=10), the native leaflets were removed before similar valve deployment. Coronary flow measurements were repeated post-implantation. RESULTS: In Group A, valve implantation resulted in a significant decrease in both left and right coronary flows. In Group B, no significant change in either right or left coronary flow was found after valve placement. CONCLUSION: Implantation of a percutaneous valved stent in the orthotopic position with the native valve in place causes coronary ostial obstruction. This problem highlights the need for modified stents that are designed for implantation in patients with non-retracted, fibrotic, or calcified leaflets.

Dimensional ratios of normal mitral valve structures: a tool for determining the degree of geometric distortion in individual patients.

BACKGROUND AND AIM OF THE STUDY: One objective of mitral valve repair is to restore the distorted mitral apparatus geometry to its normal dimensions specific for each patient. Because all dimensions of the normal aortic and mitral valves should be related, it was hypothesized that, in the presence of a normal aortic annulus, it would be possible to determine the dimensions of the structures needed for mitral valve repair. METHODS: In seven sheep, sonometric ultrasound crystals were implanted at the left and right trigones (T1, T2), lateral annulus (P1, P2), and the tips of the anterior and posterior papillary muscles (Ml, M2). The distances T1-T2, M1-M2, T1-M1, T2-M2, P1-P2, P1-M1, and P2-M2 were measured at end-systole (ES), end-diastole (ED), and maximum and minimum lengths. Using these measured distances, fractional relationships were computed, and the average fractional relationship was used to determine a 'calculated' distance. The 'measured' and 'calculated' distances were then compared using a paired t-test. RESULTS: All fractional relationships were close to 1, with ED 1.00 +/- 0.21, ES 0.99 +/- 0.19, maximum length 0.99 +/- 0.19, and minimum length 0.94 +/- 0.21. The intertrigonal distance (T1-T2) expanded by 4.19 +/- 3.81%, and the transverse diameter (P1-P2) contracted by -6.15 +/- 3.69% from ED to ES. The interpapillary muscle distance (M1-M2) contracted -22.3 +/- 6.5%. The two distances with the least amount of contraction were those of T1-M1 and T2-M2, with contractions of -3.06 +/- 2.39% and -3.27 +/- 1.37%, respectively. P1-M1 and P2-M2 expanded 5.60 +/- 2.89% and 6.84 +/- 3.60% from ED to ES. CONCLUSION: The mitral valve dimensions and calculated fractional relationships were similar in all sheep. As shown previously, the ratio of aortic annulus diameter (easily measured echocardiographically) to the intertrigonal distance (T1-T2) is 0.79 and 0.80 in humans and sheep, respectively. This distance can be used to determine normal mitral valve geometry and, therefore, preoperatively to calculate the degree of geometric distortion present in individual patients.

The papillary muscles as shock absorbers of the mitral valve complex. An experimental study.

OBJECTIVE: Although it is known that the papillary muscles ensure the continuity between the left ventricle (LV) and the mitral apparatus, their precise mechanism needs further study. We hypothesize that the papillary muscles function as shock absorbers to maintain a constant distance between their tips and the mitral annulus during the entire cardiac cycle. MATERIALS AND METHODS: Sonomicrometry crystals were implanted in five sheep in the mitral annulus at the trigones (T1 and T2), mid anterior annulus (AA) mid posterior annulus (PA), base of the posterior lateral scallops (P1 and P2), tips of papillary muscles (M1 and M2), and LV apex. LV and aortic pressures were simultaneously recorded and used to define the different phases of the cardiac cycle. RESULTS: No significant distance changes were found during the cardiac cycle between each papillary muscle tip and their corresponding mitral hemi-annulus: M1-T1, (3.5+/-2%); M1-P1 (5+/-2%); M1-PA (5+/-3%); M2-T2 (2.7+/-2%); M2-P2 (6.1+/-3%); and M2-AA (4.2+/-3%); (p>0.05, ANOVA). Significant changes were observed in distances between each papillary muscle tip and the contralateral hemi-mitral annulus: M1-T2 (1.7+/-3%); M1-P2 (23+/-6%); M1-AA (6+/-3%); M2-T1 (8+/-3%); M2-P1 (10.5+/-6%); and M2-PA (12.6+/-8%); (p<0.05 ANOVA). The distance changes between LV apex and each papillary muscle tip were significantly different: apex-M1 (12.9+/-1%) and apex-M2 (10.5+/-1%) and different from the averaged distance change between the LV apex and each annulus crystal (8.3+/-1%) with p<0.05. CONCLUSION: The papillary muscles seem to be independent mechanisms designed to work as shock absorbers to maintain the basic mitral valve geometry constant during the cardiac cycle.

Sutureless stented aortic valve implantation under direct vision: lessons from a negative experience in sheep.

BACKGROUND AND AIM OF THE STUDY: Percutaneous aortic valve replacement has been proposed as a valid alternative to surgery in selected cases; however, it still has many problems. As a less radical preliminary step, we implanted a balloon-expandable stented aortic valve under direct vision in sheep. METHODS: Under cardiopulmonary bypass (CPB) and through a transverse aortotomy, an aortic valve mounted in a long tubular balloon-expandable stent was implanted in six acute sheep. The leaflets were not excised and no anchoring sutures were used between stent and native annulus. Epicardial, two-dimensional color Doppler echocardiography was used to assess the function of the stented valve followed by macroscopic inspection at necropsy. RESULTS: Direct visualization of the entire annulus when the collapsed, valved stent was placed within the aortic root was difficult in all animals. Valve deployment took less than 1 minute. The surgical procedure resulted in major complications in all cases. Migration (3/6), paravalvular leak (2/6), mitral conflicts resulting in mitral regurgitation (1/6), and coronary ostia obstruction (2/6) were the major events at the origin of the failure. Only three animals could be weaned from CPB but did not recover enough to survive the procedure. CONCLUSIONS: Sutureless implantation of a stented aortic valve through standard CPB and aortotomy is far more complex than expected. Changes in stent design and surgical approach are indicated.

Evidence of mitigated calcification of the Mosaic versus Hancock Standard valve xenograft in the mitral position of young sheep.

OBJECTIVES: Durability remains the main problem of all bioprosthetic valves, and calcification is the major cause of failure. New tissue treatment processes are expected to reduce mineralization. A comparative animal study was undertaken to evaluate the behavior of a new-generation porcine bioprosthesis in contrast with a first-generation porcine bioprosthesis. The primary goal was to evaluate the efficacy of alpha-amino-oleic acid as an anticalcification treatment. METHODS: Seventeen Targhee sheep (aged 4.5-7 months) had a mitral valve replacement with a Mosaic or Hancock Standard. The animals were followed up to 20 weeks (144.1 +/- 4.0 days vs 144.3 +/- 8.2 days) and then euthanized as scheduled. After gross examination, the explants were radiographed for the presence of calcification. The central portions were preserved for histologic examination, and the remainder of the sample was analyzed for quantitative calcium content by atomic absorption spectroscopy. RESULTS: Four Mosaic sheep were excluded because of perioperative surgical mortality. The remaining 13 were enrolled in the study (9 Mosaic and 4 Hancock Standard). The mean calcium content was 1.97 +/- 2.21 microg/mg tissue weight for Mosaic versus 8.36 +/- 4.12 microg/mg for Hancock Standard valves (P < .01). Mild fibrous tissue overgrowth and fibrinous lining were observed regardless the xenograft type. CONCLUSIONS: The low level of calcification in the Mosaic versus Hancock Standard xenografts confirms the efficacy of alpha-amino-oleic acid treatment in mitigating mineralization. A longer durability is expected with the clinical use of the Mosaic porcine valve.

Kinking of the atrioventricular plane during the cardiac cycle.

Systolic descent of the atrioventricular plane toward the relatively stationary left ventricular apex is well described. As the atrioventricular plane includes two separate valvular units, systolic atrioventricular plane displacement should not be homogenous. In 6 sheep, sonomicrometric crystals were implanted at the base of the right coronary sinus, anterolateral and posteromedial fibrous trigones, posterior mitral annulus, left ventricular apex, and the tips of the anterior and posterior mitral leaflets. The aortomitral angle was calculated and related to simultaneous left ventricular and aortic pressures and mitral valve movement. The aortomitral angle was largest at end diastole (150.73 degrees +/- 15.48 degrees ). During isovolumic contraction, it narrowed rapidly to 144.90 degrees +/- 16.64 degrees , followed by a slower narrowing during ejection until it reached its smallest angle at end systole (139.66 degrees +/- 16.78 degrees ). During isovolumic relaxation, the aortomitral angle increased to 143.66 degrees +/- 16.02 degrees at the beginning of diastole. During the first third of diastole, it narrowed again to 141 degrees +/- 16.24 degrees before re-expanding to maximum at end diastole. During systole, the atrioventricular plane descended non-homogeneously toward the apex, with kinking at the hinge between the aortic and mitral annulus plane. This deformation of the atrioventricular plane has relevance in valve surgery.

An experimental method for the percutaneous induction of a posterolateral infarct and functional ischemic mitral regurgitation.

BACKGROUND AND AIM OF THE STUDY: Appropriate experimental models are needed to study the mechanisms underlying left ventricular (LV) remodeling and functional ischemic mitral regurgitation (IMR). Herein is described an original percutaneous method for inducing a well-defined posterolateral infarct and significant IMR. METHODS: Under videofluoroscopic guidance, the second (OM2) and third (OM3) obtuse marginal branches of the circumflex artery of six sheep were selectively and sequentially injected with 100% ethyl alcohol. Transthoracic echocardiography (TTE) was performed before and after alcohol injection, and weekly until sacrifice at 8 +/- 1.3 weeks. The LV endsystolic (LVESD) and end-diastolic (LVEDD) dimensions, interpapillary distance (M1-M2), mitral annulus diameter (MA), and degree of IMR and ischemic tricuspid regurgitation (ITR) were measured. RESULTS: One animal died from irreversible ventricular fibrillation. In the remaining sheep, a well-defined posterolateral infarct of 22% of the heart mass resulted, followed by 2.8 + IMR and 2.1+ ITR. The mean weight gain was 16%, and all sheep showed signs of heart failure. All echocardiographic parameters were increased: systolic MA by 29%, diastolic MA by 18%, LVEDD by 33%, LVESD by 62%, M1-M2 diastolic by 32%, M1-M2 systolic by 21%, and tethering and tenting distances by 32% and 108%, respectively. CONCLUSION: The percutaneous selective injection of 100% ethyl alcohol in OM2 and OM3 resulted in a well-defined posterolateral infarct and significant IMR and ITR. Because it was a percutaneous procedure, this novel, simple and reproducible method did not require a thoracotomy. This model should facilitate the further study of LV remodeling and IMR.

Anterior mitral basal 'stay' chords are essential for left ventricular geometry and function.

BACKGROUND AND AIM OF THE STUDY: Among the anterior mitral basal chords, two particularly strong and thick stay chords (SC) remain under tension during the entire cardiac cycle. Collagen fibers of the anterior mitral leaflet (AML) are oriented from insertion of the SC on the AML to the fibrous trigones (FT), suggesting that local stress is directed from the papillary muscles (PM) over the SC and AML to the FT, maintaining left ventricular (LV) geometry. METHODS: Sonomicrometry crystals were implanted in sheep at the LV apex (A), the anterior (AW) and septal (SW) LV wall, the PM tips (M1 and M2), the SC insertion into the AML (S1 and S2), the posterior (PMA) and lateral (P1 and P2) mitral annulus, the FT (T1 and T2), the tips of the anterior (AL) and posterior (PL) mitral leaflets, and the base of the aortic right coronary sinus (RCS). Changes in distances, areas, and volume were time-related to aortic flow and LV and ascending aorta pressures. Recordings were taken at baseline and after transection of the SC. RESULTS: After transection of the SC, the systolic distance from M1-T1 increased by +0.96 +/- 0.41 mm (p < 0.05) and from M2-T2 by +0.97 +/- 0.42 mm (p < 0.05). The LV length increased at T1-A by +1.14 +/- 0.60 mm (p < 0.05) and at T2-A by +0.97 +/- 0.37 mm (p < 0.05). The aortomitral angle narrowed at end-systole by -3.26 +/- 0.85 degrees (p < 0.05). Transection of the SC reduced dP/dt by -11.20 +/- 5.29% (p < 0.05), maximum aortic flow by -16.89 +/- 7.86% (p < 0.05), and maximum pressure-volume ratio by -10.83 +/- 3.36% (p < 0.05). CONCLUSION: Transection of the anterior mitral SC did not result in mitral regurgitation but induced significant changes in LV geometry, including narrowing of the aortomitral angle and subsequent deterioration of LV function. The SC are essential for maintaining normal LV geometry and function.

Left ventricular endocardial longitudinal and transverse changes during isovolumic contraction and relaxation: a challenge.

Left ventricular (LV) longitudinal and transverse geometric changes during isovolumic contraction and relaxation are still controversial. This confusion is compounded by traditional definitions of these phases of the cardiac cycle. High-resolution sonomicrometry studies might clarify these issues. Crystals were implanted in six sheep at the LV apex, fibrous trigones, lateral and posterior mitral annulus, base of the aortic right coronary sinus, anterior and septal endocardial wall, papillary muscle tips, and edge of the anterior and posterior mitral leaflets. Changes in distances were time related to LV and aortic pressures and to mitral valve opening. At the beginning of isovolumic contraction, while the mitral valve was still open, the LV endocardial transverse diameter started to shorten while the endocardial longitudinal diameter increased. During isovolumic relaxation, while the mitral valve was closed, LV transverse diameter started to increase while the longitudinal diameter continued to decrease. These findings are inconsistent with the classic definitions of the phases of the cardiac cycle.

Halofuginone inhibits serum-stimulated pericardial tissue retraction in vitro.

We developed an in vitro model of tissue contraction in which living pericardium, in response to serum, contracted and the cells in situ expressed proliferating cell nuclear antigen (PCNA) and synthesized collagen. Here we evaluated the effects of halofuginone on these serum-stimulated pericardial tissue responses. Parietal pericardium was incubated with media containing increasing doses of halofuginone and evaluated for tissue contraction, evident by tissue curling. Proliferation was measured by MTS metabolism and PCNA expression. Furthermore, collagen synthesis was compared between samples incubated with halofuginone, cytochalasin B, cytochalasin D, aphidicolin, or cytosine arabinoside (AraC), using Masson's trichrome and the monoclonal antibody to sheep type I procollagen, SP1. D8. Halofuginone inhibited tissue contraction, cellular proliferation, and collagen synthesis in a dose-dependent manner. In contrast, cytochalasin B, cytochalasin D, aphidicolin, and AraC, shown previously to prevent cellular proliferation, did not prevent type I collagen synthesis. Halofuginone has been implicated as an agent in the prevention of wound-healing fibrosis. This study suggests that halofuginone may have an added benefit in the inhibition of pericardial tissue contraction, which appeared to be related to the synthesis of type I procollagen.

Improving the results of small-bore vascular graft testing in rabbits: a technical primer.

Appropriate models to evaluate the in vivo behavior of small-diameter grafts are varied. To evaluate the behavior of small-diameter, bovine-derived grafts in the arterial circulation, we chose the rabbit abdominal aorta model. In the development of our procedure, we evaluated several models published in the literature, with unsatisfactory results. The high incidence of postoperative mortality and morbidity led us to modify published methods to incorporate cautious surgical technique and mild systemic hypothermia with cross-clamp times shorter than 30 min, as well as perioperative administration of agents with metabolic, rheologic, and neuroprotective properties. These modifications enabled us to achieve 100% operative survival with a very low incidence of postoperative paralysis. The presented model will be used for further evaluation of small-diameter grafts in our laboratory.

Anterior mitral leaflet mobility is limited by the basal stay chords.

BACKGROUND: We hypothesize that 2 tendon-like anterior basal stay chords, which remain taut during the entire cardiac cycle, limit the motion of the anterior mitral leaflet. METHODS AND RESULTS: Sonomicrometric crystals were implanted in 6 sheep at the insertion of stay chords at anterior mitral leaflet (S1 and S2), papillary muscle tips, fibrous trigones, mitral annulus, and the tip of the anterior leaflet (AL). Distances between crystals were recorded before and after section of stay chords. During the cardiac cycle, the angle alpha between mitral annulus and AL changed by +54.2+/-12.4 degrees; the angles between mitral annulus and S1 (beta1) changed by +25.7+/-14.6 degrees, and between mitral annulus and S2 (beta2) by +20.4+/-7.8 degrees. During diastole, AL twice crossed the virtual plane formed by the stay chords: during E-wave by a maximum of 6.5 mm (mean, 2.5+/-2.2 mm) and during A-wave by a maximum of 3.2 mm (mean, 1.7+/-0.9 mm). After section of both stay chords, total anterior mitral leaflet motion increased as follows: AL, +6.9+/-3.4 degrees; S1, +13.1+/-4.4 degrees; and S2, +30.9+/-11.7 degrees (P<0.05). CONCLUSIONS: Although the lateral movement of anterior mitral leaflet is limited by stay chords, the midportion moves unimpaired toward the septum, like a sail, between the 2 stay chords during diastole. A diastolic left ventricular-inflow and systolic left ventricular-outflow funnel mechanism is created. Stay chord section increased lateral anterior mitral leaflet movement.

Cellular expression of PCNA and procollagen in vital and ethanol-treated autologous pericardial implants in sheep.

BACKGROUND AND AIMS OF THE STUDY: Cardiovascular surgeries involving repair or reconstruction of heart valve leaflets with vital autologous pericardium have shown detrimental healing outcomes, mainly fibrosis with retraction. It is proposed that cells intrinsic to the pericardial implants may contribute to this fibrosis by becoming activated to proliferate and synthesize type I collagen. METHODS: Vital and ethanol-treated autologous pericardium were implanted as rectangular flaps bisecting the lumen in the descending aorta of sheep to simulate a heart valve leaflet. Implants recovered at 5, 10, 15, and 30 days were evaluated immunohistologically for expression of PCNA and procollagen. RESULTS: In ethanol-treated pericardium, concentrations of activated cells shifted from the fibrin layers on the periphery of implants at days 5 and 10 to cells internal to the implant at days 15 and 30. In contrast, concentrations of activated cells in vital pericardium shifted from cells within the implants at days 5 and 10 to the fibrin deposits overlaying the implants at days 15 and 30. CONCLUSION: Different distributional patterns of activated cells were observed between vital and ethanol-treated pericardial flap implants. These different patterns may be important in understanding the cause of the detrimental healing outcome observed with vital autologous pericardial flap implants.

An in vitro model of pericardial tissue healing.

INTRODUCTION: A previous study in our laboratory showed that a flap of fresh autologous pericardium bisecting the aorta of sheep retracted and became fibrotic. Histologic analyses suggested that activated cells within the pericardium contributed to the retraction of the implant. Here we report the development of an in vitro model to investigate the effects of serum on cellular proliferation and cell-mediated tissue contraction. METHODS: Sections of living and ethanol-treated sheep pericardium were incubated with 0.5%, 5%, 10%, 20%, and 50% serum in medium for up to 8 days and evaluated for cellular proliferation and tissue contraction. These serum-stimulated events were further evaluated in the presence of Mitomycin C, Cytochalasin B and D, Aphidicolin, AraC, and Cycloheximide. RESULTS: Cellular proliferation and cell-mediated tissue contraction were induced by serum in a dose-dependent manner. Expression of PCNA was suppressed in the presence of Cytochalasin B, Cytochalasin D, Aphidicolin, and AraC. Tissue contraction was prevented by Cycloheximide. Mitomycin C inhibited both proliferation and tissue contraction. Ethanol-treated tissue, which was absent of living cells, did not respond to stimulation with serum. CONCLUSIONS: An in vitro model was developed to study the responses of cells within pericardial tissues to stimulation by serum. In this model, serum induced cellular proliferation and tissue contraction. Different chemical inhibitors independently modulated these serum-stimulated events. Pre-existing cells within pericardial tissues might respond to stimulus through differential pathways. This model may help to develop methods to make autologous pericardium a clinically useful biomaterial.

A temporarily stented, autologous pericardial aortic valve prosthesis.

BACKGROUND AND AIM OF THE STUDY: A new bioprosthesis has been developed that is: (i) constructed from glutaraldehyde-treated autologous pericardium due to its lack of antigenicity and low cost; (ii) easily constructed in the operating room; (iii) stentless but easy to implant by using a temporary stent that is removed once its function is no longer necessary; and (iv) implanted with a single proximal suture and three commissural stitches. METHODS: This prosthesis was implanted in the subcoronary aortic position of six sheep. The mean prosthesis size was 19.7 +/- 3.5 mm, and manufacturing time 16 +/- 3 min. Cardiopulmonary bypass and cross-clamp times were 142.5 +/- 26.7 min and 100.2 +/- 28.8 min, respectively. Three sheep were euthanized at three, seven, or 27 days, and the remainder at five months postoperatively. RESULTS: In all cases the valve was competent and the single commissural stitches were well-anchored. The pericardium was pliable in all cases, but one animal had obvious endocarditis (at 27 days). Small calcific nodules were found at the commissures in valves explanted at five months. Histology showed an intact collagen and elastin structure, and neointimal growth was seen covering the basal quarter of the leaflets and commissures. CONCLUSION: This new bioprosthesis might offer a particularly attractive alternative for the young patient population of the developing world. The simplicity of construction, low cost, and absence of any need for anticoagulation makes this prosthesis close to the ideal aortic valve substitute.

Serum components stimulate pericardial tissue contraction.

BACKGROUND AND AIMS OF THE STUDY: Previous experiments have demonstrated the retraction and fibrosis of vital autologous pericardial flap implants in the descending aorta of sheep. An in-vitro model of pericardial tissue contraction was developed that showed healing reactions similar to those observed in the fresh in-vivo flap. Here, the component(s) of serum that stimulate tissue contraction were partially characterized. The molecular weight range and stability (heat and protease resistance) of the serum component(s) are described. Tissue contraction also requires de-novo protein synthesis. METHODS: Sections (1 cm2) of sheep pericardium were incubated with fractionated, heat-treated, or protease-treated fetal bovine serum for 14 days. In addition, SDS-PAGE protein profiles were generated using tissues incubated with and without cycloheximide for up to 12 days. RESULTS: Tissue contraction was observed in molecular weight serum fractions > or =5 kDa, as well as in samples incubated with heat and protease-treated serum. SDS-PAGE showed the appearance of a protein band after day 4 during the process of tissue contraction that was absent in samples incubated with cycloheximide. CONCLUSION: Serum fractions > or =5 kDa stimulated protein synthesis and pericardial tissue contraction. The active component(s) was shown to be heat stable, but partially sensitive to protease. The addition of cycloheximide to the culture medium, shown previously to prevent pericardial tissue contraction, inhibited de-novo synthesis of the protein that appeared during the process of tissue contraction.