Tissue-engineered vascular grafts transform into mature blood vessels via an inflammation-mediated process of vascular remodeling.

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are the earliest tissue-engineered vascular grafts (TEVGs) to be used clinically. These TEVGs transform into living blood vessels in vivo, with an endothelial cell (EC) lining invested by smooth muscle cells (SMCs); however, the process by which this occurs is unclear. To test if the seeded BMCs differentiate into the mature vascular cells of the neovessel, we implanted an immunodeficient mouse recipient with human BMC (hBMC)-seeded scaffolds. As in humans, TEVGs implanted in a mouse host as venous interposition grafts gradually transformed into living blood vessels over a 6-month time course. Seeded hBMCs, however, were no longer detectable within a few days of implantation. Instead, scaffolds were initially repopulated by mouse monocytes and subsequently repopulated by mouse SMCs and ECs. Seeded BMCs secreted significant amounts of monocyte chemoattractant protein-1 and increased early monocyte recruitment. These findings suggest TEVGs transform into functional neovessels via an inflammatory process of vascular remodeling.

Correlation between body weight changes and postoperative pain in rats treated with meloxicam or buprenorphine.

It is essential to identify objective and efficient methods of evaluating postoperative pain in rodents. The authors investigated whether postoperative changes in rates of body weight gain could serve as a measure of the efficacy of meloxicam or buprenorphine analgesia in growing rats. Young adult male Lewis rats underwent general endotracheal anesthesia and thoracotomy and were treated postoperatively for 3 d with saline (no analgesia), buprenorphine (six doses of 0.1 mg per kg) or meloxicam (three doses of 1 mg per kg). The authors evaluated rats' daily growth rates for 5 d after surgery and compared them with baseline (preoperative) growth rates. To discriminate between the effects of postoperative pain and other concurrent physiologic effects associated with anesthesia, thoracotomy or analgesia, the authors evaluated weight changes in multiple control groups. Treatment with buprenorphine in the absence of any other procedure or with anesthesia alone significantly affected rats' body weight. Notably, growth rate was maintained at near normal levels in rats treated postoperatively with meloxicam. These findings suggest that growth rate might serve as an efficient index of postoperative pain after major surgical procedures in young adult rats treated with meloxicam but not in rats treated with buprenorphine.

Tissue-engineered vascular grafts demonstrate evidence of growth and development when implanted in a juvenile animal model.

INTRODUCTION: The development of a living, autologous vascular graft with the ability to grow holds great promise for advancing the field of pediatric cardiothoracic surgery. OBJECTIVE: To evaluate the growth potential of a tissue-engineered vascular graft (TEVG) in a juvenile animal model. METHODS: Polyglycolic acid nonwoven mesh tubes (3-cm length, 1.3-cm id; Concordia Fibers) coated with a 10% copolymer solution of 50:50 L-lactide and epsilon-caprolactone were statically seeded with 1 x 10 cells/cm autologous bone marrow derived mononuclear cells. Eight TEVGs (7 seeded, 1 unseeded control) were implanted as inferior vena cava (IVC) interposition grafts in juvenile lambs. Subjects underwent bimonthly magnetic resonance angiography (Siemens 1.5 T) with vascular image analysis (www.BioimageSuite.org). One of 7-seeded grafts was explanted after 1 month and all others were explanted 6 months after implantation. Neotissue was characterized using qualitative histologic and immunohistochemical staining and quantitative biochemical analysis. RESULTS: All grafts explanted at 6 months were patent and increased in volume as measured by difference in pixel summation in magnetic resonance angiography at 1 month and 6 months. The volume of seeded TEVGs at explant averaged 126.9% +/- 9.9% of their volume at 1 month. Magnetic resonance imaging demonstrated no evidence of aneurysmal dilation. TEVG resembled the native IVC histologically and had comparable collagen (157.9 +/- 26.4 microg/mg), elastin (186.9 +/- 16.7 microg/mg), and glycosaminoglycan (9.7 +/- 0.8 microg/mg) contents. Immunohistochemical staining and Western blot analysis showed that Ephrin-B4, a determinant of normal venous development, was acquired in the seeded grafts 6 months after implantation. CONCLUSIONS: TEVGs demonstrate evidence of growth and venous development when implanted in the IVC of a juvenile lamb model.

Small-diameter biodegradable scaffolds for functional vascular tissue engineering in the mouse model.

The development of neotissue in tissue engineered vascular grafts remains poorly understood. Advances in mouse genetic models have been highly informative in the study of vascular biology, but have been inaccessible to vascular tissue engineers due to technical limitations on the use of mouse recipients. To this end, we have developed a method for constructing sub-1mm internal diameter (ID) biodegradable scaffolds utilizing a dual cylinder chamber molding system and a hybrid polyester sealant scaled for use in a mouse model. Scaffolds constructed from either polyglycolic acid or poly-l-lactic acid nonwoven felts demonstrated sufficient porosity, biomechanical profile, and biocompatibility to function as vascular grafts. The scaffolds implanted as either inferior vena cava or aortic interposition grafts in SCID/bg mice demonstrated excellent patency without evidence of thromboembolic complications or aneurysm formation. A foreign body immune response was observed with marked macrophage infiltration and giant cell formation by post-operative week 3. Organized vascular neotissue, consisting of endothelialization, medial generation, and collagen deposition, was evident within the internal lumen of the scaffolds by post-operative week 6. These results present the ability to create sub-1mm ID biodegradable tubular scaffolds that are functional as vascular grafts, and provide an experimental approach for the study of vascular tissue engineering using mouse models.

Centrifugal seeding increases seeding efficiency and cellular distribution of bone marrow stromal cells in porous biodegradable scaffolds.

Bone marrow stromal cells (MSCs) are a promising cell source for a variety of tissue engineering applications, given their ready availability and ability to differentiate into multiple cell lineages. MSCs have been successfully used to create neotissue for cardiovascular, urological, and orthopedic reconstructive surgical procedures in preclinical studies. The ability to optimize seeding techniques of MSCs onto tissue engineering scaffolds and the ability to control neotissue formation in vitro will be important for the rational design of future tissue engineering applications using MSCs. In this study we investigated the effect of centrifugal force on seeding MSCs into a biodegradable polyester scaffold. MSCs were isolated and seeded onto porous scaffold sections composed of nonwoven polyglycolic acid mesh coated with poly(L-lactide-co-epsilon-caprolactone). Compared to standard static seeding techniques, centrifugal seeding increased the seeding efficiency by 38% (p < 0.007) and significantly improved cellular distribution throughout the scaffold. Overall, centrifugal seeding of MSCs enhances seeding efficiency and improves cellular penetration into scaffolds, making it a potentially useful technique for manipulating neotissue formation by MSCs for tissue engineering applications.

Accuracy and reproducibility of absolute quantification of myocardial focal tracer uptake from molecularly targeted SPECT/CT: a canine validation.

UNLABELLED: Accurate and reproducible SPECT quantification of myocardial molecular processes remains a challenge because of the complication of heterogeneous background and extracardiac activity adjacent to the heart, which causes errors in the estimation of myocardial focal tracer uptake. Our aim in this study was to introduce a heuristic method for the correction of extracardiac activity into SPECT quantification and validate the modified quantification method for accuracy and reproducibility using a canine model. METHODS: Dual-isotope-targeted (99m)Tc and (201)Tl perfusion SPECT images were acquired using a hybrid SPECT/CT camera in 6 dogs at 2 wk after myocardial infarction. Images were reconstructed with and without CT-based attenuation correction, and the reconstructed SPECT images were filtered and quantified simultaneously with incorporation of extracardiac radioactivity correction, gaussian fitting, and total-count sampling. Absolute myocardial focal tracer uptake was quantified from SPECT images using 3 different normal limits (maximum entropy [ME], mean-squared-error minimization [MSEM], and global minimum [GM]). SPECT-quantified percentage injected dose (%ID) was calculated and compared with the well-counted radioactivity measured from the postmortem myocardial tissue. SPECT quantitative processing was performed by 2 different individuals with extensive experience in cardiac image processing, to assess reproducibility of the quantitative analysis. RESULTS: Correlations between SPECT-quantified and well-counted %IDs using 3 different normal limits were excellent (ME: r = 0.82, y = 0.932 x - 0.0102; MSEM: r = 0.73, y = 1.1413 x - 0.0052; and GM: r = 0.7, y = 1.2147 x - 0.0002). SPECT quantification using ME normal limits resulted in an underestimation of %ID, as compared with well-counted %ID. Myocardial focal tracer uptake quantified from SPECT images without CT-based attenuation correction was significantly lower than that with the attenuation correction. The %IDs quantified from attenuation-corrected SPECT images using MSEM and GM normal limits were not significantly different from well-counted %IDs. Reproducibility of the SPECT quantitative analysis was excellent (ME: r = 0.98, y = 0.9221 x + 0.0001; MSEM: r = 0.97, y = 0.9357 x + 0.0004; and GM: r = 0.96, y = 0.9026 x + 0.001). CONCLUSION: Our SPECT/CT quantification algorithm for the assessment of regional radioactivity may allow for accurate and reproducible serial noninvasive evaluation of molecularly targeted tracers in the myocardium.

A non-parametric vessel detection method for complex vascular structures.

Modern medical imaging techniques enable the acquisition of in vivo high resolution images of the vascular system. Most common methods for the detection of vessels in these images, such as multiscale Hessian-based operators and matched filters, rely on the assumption that at each voxel there is a single cylinder. Such an assumption is clearly violated at the multitude of branching points that are easily observed in all, but the most focused vascular image studies. In this paper, we propose a novel method for detecting vessels in medical images that relaxes this single cylinder assumption. We directly exploit local neighborhood intensities and extract characteristics of the local intensity profile (in a spherical polar coordinate system) which we term as the polar neighborhood intensity profile. We present a new method to capture the common properties shared by polar neighborhood intensity profiles for all the types of vascular points belonging to the vascular system. The new method enables us to detect vessels even near complex extreme points, including branching points. Our method demonstrates improved performance over standard methods on both 2D synthetic images and 3D animal and clinical vascular images, particularly close to vessel branching regions.

Tissue-engineered arterial grafts: long-term results after implantation in a small animal model.

BACKGROUND: Use of prosthetic vascular grafts in pediatric vascular surgical applications is limited because of risk of infection, poor durability, potential for thromboembolic complications, and lack of growth potential. Construction of an autologous neovessel using tissue engineering technology offers the potential to create an improved vascular conduit for use in pediatric vascular applications. METHODS: Tissue-engineered vascular grafts were assembled from biodegradable tubular scaffolds fabricated from poly-L-lactic acid mesh coated with epsilon-caprolactone and L-lactide copolymer. Thirteen scaffolds were seeded with human aortic endothelial and smooth muscle cells and implanted as infrarenal aortic interposition grafts in SCID/bg mice. Grafts were analyzed at time-points ranging from 4 days to 1 year after implantation. RESULTS: All grafts remained patent without evidence of thromboembolic complications, graft stenosis, or graft rupture as documented by serial ultrasound and computed tomographic angiogram, and confirmed histologically. All grafts demonstrated extensive remodeling leading to the development of well-circumscribed neovessels with an endothelial inner lining, neomedia containing smooth muscle cells and elastin, and a collagen-rich extracellular matrix. CONCLUSIONS: The development of second-generation tissue-engineered vascular grafts shows marked improvement over previous grafts and confirms feasibility of using tissue engineering technology to create an improved arterial conduit for use in pediatric vascular surgical applications.

Functional small-diameter human tissue-engineered arterial grafts in an immunodeficient mouse model: preliminary findings.

HYPOTHESIS: The immunodeficient (severe combined immunodeficiency beige [SCID/bg]) mouse model provides a useful model for investigating vascular neotissue formation in human tissue-engineered arterial conduits (TEAC). DESIGN: Human aortic smooth muscle cells and endothelial cells were statically seeded on porous biodegradable polymeric scaffolds for vascular tissue engineering. These 2-cell tissue-engineered vascular conduits were implanted into immunodeficient female mice as aortic interposition grafts. Grafts were evaluated over a 30-week course to investigate their patency and structure. SETTING: In vivo animal study. PATIENTS: Thirteen female C.B-17 SCID/bg mice. INTERVENTION: The TEACs implanted as infrarenal abdominal aortic interposition grafts. MAIN OUTCOME MEASURES: Selective microcomputed tomography with intra-arterial contrast revealed graft patency and structure. Histological and immunohistochemical evaluations revealed cellularity and extracellular matrix composition. Species-specific immunohistochemical evaluation determined the source of cells within TEACs. RESULTS: All TEACs were patent without evidence of thrombosis or rupture over the 30-week course. Histological and immunohistochemical evaluation revealed a von Willebrand factor-positive luminal monolayer surrounded by concentric collagen-rich layers of alpha-smooth muscle actin-positive cells. CONCLUSIONS: The SCID/bg mouse is a useful model for investigating vascular neotissue formation in human TEACs. We see evidence that these grafts remain patent while developing into vascular neotissue histologically similar to native aorta. This chimeric animal model also enables determination of seeded cell retention, providing insight into cellular mechanisms underlying neotissue formation.

Development of a mouse model for evaluation of small diameter vascular grafts.

BACKGROUND: It is estimated that 80,000 individuals are unable to undergo life or limb saving bypass surgery because of inadequate small caliber synthetic vascular grafts. The use of tissue engineering methods has been proposed as a potential means of creating improved vascular conduits. We have developed a severe combined immunodeficiency (SCID) mouse aortic interposition model for initial evaluation and screening of small diameter vascular conduits in vivo. METHODS: Fifteen small diameter vascular conduits, approximately 1 mm in diameter and 10 mm in length, were implanted as infrarenal aortic interposition grafts for 1 to 35 days. Eight grafts were constructed using a decellularized ovine arterial tissue as a scaffold. Seven grafts were constructed from silastic tubing. Four grafts were composed of Polyurethane, and two were made of expanded polytetrafluroethylene. To explore noninvasive means of evaluating patency, grafts were selectively imaged using ultrasound and micro-computed tomography. RESULTS: All grafts were patent immediately post-operatively and at time of sacrifice. All imaging modalities were able to visualize the grafts and confirm patency. All specimens were sent for histology to evaluate neotissue formation and to correlate radiographic morphology with histological morphology. CONCLUSIONS: The use of the SCID mouse model for initial evaluation of small caliber grafts is feasible and provides a cost effective rapid screening model with the added advantage of being able to use human cells in further studies.

Construction of an autologous tissue-engineered venous conduit from bone marrow-derived vascular cells: optimization of cell harvest and seeding techniques.

BACKGROUND: Currently available vascular grafts for pediatric cardiovascular operations are limited by their inability to grow. Tissue-engineering techniques can be used to create vascular grafts with the potential for repair, remodeling, and growth. This study demonstrates the feasibility of constructing an autologous tissue-engineered venous conduit from bone marrow-derived vascular cells (BMVCs) in the ovine animal model. METHODS: Ovine mononuclear cells were isolated from the bone marrow, cultured in endothelial growth medium, and characterized with immunocytochemistry. Biodegradable tubular scaffolds were constructed from polyglycolic acid mesh coated with a copolymer of poly[epsilon-caprolactone-L-lactide]. Scaffolds were seeded at various cell concentrations and incubation times to optimize seeding conditions for the construction of an autologous venous conduit. Using optimized conditions, 6 tissue-engineered vascular grafts were implanted as inferior vena cava interposition grafts in juvenile lambs. Grafts were assessed for patency at days 1 to 30 postoperatively and explanted for histological and immunohistochemical analysis. RESULTS: A mixed cell population of BMVCs consisting of smooth muscle cells and endothelial cells was cultured from ovine sternal bone marrow. A seeding concentration of 2 x 10(6) cells/cm2 and 7 days of postseeding incubation were optimal for creating a confluent cellular layer on the polyglycolic acid/poly[epsilon-caprolactone-L-lactide]) scaffold. Grafts were explanted up to 4 weeks postoperatively. All grafts were patent without evidence of thrombosis. Histological evaluation of the explanted grafts demonstrated neo-endothelialization. Graft wall was composed of neo-tissue made up of residual polymer matrix, mesenchymal cells, and extracellular matrix without evidence of calcification. CONCLUSIONS: Bone marrow-derived vascular cells, containing endothelial and smooth muscle cells, can be isolated and cultured from ovine sternal bone marrow and used as a cell source for vascular tissue engineering. Our optimized techniques for BMVC harvest and seeding onto biodegradable scaffolds can be used for studying autologous tissue-engineered vascular grafts in the ovine animal model.