Combination of bone morphogenetic protein-2 plasmid DNA with chemokine CXCL12 creates an additive effect on bone formation onset and volume.

Bone morphogenetic protein-2 (BMP-2) gene delivery has shown to induce bone formation in vivo in cell-based tissue engineering. In addition, the chemoattractant stromal cell-derived factor-1α (SDF-1α, also known as CXCL12) is known to recruit multipotent stromal cells towards its release site where it enhances vascularisation and possibly contributes to osteogenic differentiation. To investigate potential cooperative behaviour for bone formation, we investigated combined release of BMP-2 and SDF-1α on ectopic bone formation in mice. Multipotent stromal cell-seeded and cell-free constructs with BMP-2 plasmid DNA and /or SDF-1α loaded onto gelatin microparticles, were implanted subcutaneously in mice for a period of 6 weeks. Histological analysis and histomorphometry revealed that the onset of bone formation and the formed bone volume were both enhanced by the combination of BMP-2 and SDF-1α compared to controls in cell-seeded constructs. Samples without seeded multipotent stromal cells failed to induce any bone formation. We conclude that the addition of stromal cell-derived factor-1α to a cell-seeded alginate based bone morphogenetic protein-2 plasmid DNA construct has an additive effect on bone formation and can be considered a promising combination for bone regeneration.

Metalloproteinase inhibition reduces constrictive arterial remodeling after balloon angioplasty: a study in the atherosclerotic Yucatan micropig.

BACKGROUND: Arterial remodeling after balloon angioplasty has been recognized as a major determinant of restenosis. Perturbation of collagen metabolism might be important. After balloon injury, matrix metalloproteinase (MMP) expression is upregulated. We investigated the effect of Batimastat, a nonspecific MMP inhibitor, on late lumen loss, arterial remodeling, and neointima formation after balloon dilation. METHODS AND RESULTS: In atherosclerotic iliac arteries of 12 Yucatan micropigs, balloon dilation was performed, with intravascular ultrasound and quantitative angiography used before and after balloon dilation and at 42-day follow-up. The animals were randomly divided into 2 groups, the Batimastat group (n=6) and the vehicle group (n=6). All animals were intraperitoneally injected with either Batimastat or a vehicle immediately after balloon dilation and at 2 weeks and 4 weeks after balloon dilation. Angiographic and echographic late lumen loss in the Batimastat group versus the vehicle group was 0.3+/-0.1 versus 0.8+/-0.1 mm (P=0.01) and 2.2+/-0.5 versus 4.9+/-0.7 mm(2) (P=0.004), respectively. Late media-bounded area loss was used as a measure of remodeling after balloon dilation and was 0.9+/-0.6 mm(2) in the Batimastat group compared with 3.8+/-0.8 mm(2) in the vehicle group (P=0.003, mixed model analysis P=0.01). Neointima formation was 1.3+/-0.3 mm(2) in the Batimastat group and 1.0+/-0.2 mm(2) in the vehicle group (P=0. 542). CONCLUSIONS: Metalloproteinase inhibition by Batimastat significantly reduced late lumen loss after balloon angioplasty by inhibition of constrictive arterial remodeling, whereas neointima formation was not inhibited by MMP inhibition.

Effects of increasing doses of simvastatin on fasting lipoprotein subfractions, and the effect of high-dose simvastatin on postprandial chylomicron remnant clearance in normotriglyceridemic patients with premature coronary sclerosis.

Postprandial hyperlipidemia has been linked to premature coronary artery disease (CAD) in fasting normotriglyceridemic patients. We investigated the effects of increasing doses of simvastatin up to 80 mg/day on fasting and postprandial lipoprotein metabolism in 18 normotriglyceridemic patients with premature CAD. Fasting lipoprotein subfractions and cholesteryl ester transfer protein (CETP) activity were determined after each 5-week dose titration (0, 20, 40 and 80 mg/day). At baseline and after treatment with simvastatin 80 mg/day, standardised Vitamin A oral fat loading tests (50 g/m2; 10 h) were carried out. Ten normolipidemic healthy control subjects matched for gender, age and BMI underwent tests without medication. Treatment with simvastatin resulted in dose-dependent reductions of fasting LDL-cholesterol, without changing cholesterol levels in the VLDL-1, VLDL-2 and IDL fractions. In addition, simvastatin decreased CETP activity dose-dependently, although HDL-cholesterol remained unchanged. Simvastatin 80 mg/day decreased fasting plasma triglycerides (TG) by 26% (P < 0.05), but did not decrease significantly TG levels in any of the subfractions. The TG/cholesterol ratio increased in all subfractions. The plasma TG response to the oral fat loading test, estimated as area under the curve (TG-AUC), improved by 30% (from 21.5 +/- 2.5 to 15.1 +/- 1.9 mmol h/L; P < 0.01). Treatment with simvastatin 80 mg/day improved chylomicron remnant clearance (RE-AUC) by 36% from 30.0 +/- 2.6 to 19.2 +/- 3.3 mg h/L (P < 0.01). After therapy, remnant clearance in patients was similar to controls (19.2 +/- 3.3 and 20.3 +/- 2.7 mg h/L, respectively), suggesting a normalization of this potentially atherogenic process. In conclusion, high-dose simvastatin has beneficial effects in normotriglyceridemic patients with premature CAD, due to improved chylomicron remnant clearance, besides effective lowering of LDL-cholesterol. In addition, the lipoprotein subfractions became more cholesterol-poor, as reflected by the increased TG/cholesterol ratio, which potentially makes them less atherogenic.

Nonmuscle myosin heavy chain-B expression in balloon-dilated and stented arteries: a study in the atherosclerotic Yucatan micropig.

BACKGROUND: Restenosis after balloon angioplasty is in part due to remodelling, whereas restenosis after stenting is entirely due to neointima formation. Nonmuscle myosin heavy chain-B (NMMHC-B) is expressed by vascular smooth muscle cells and because of its overexpression in restenotic lesions after balloon angioplasty, NMMHC-B is proposed as a potential therapeutic target. Because the mechanisms underlying restenosis after balloon angioplasty or after stenting are different we hypothesised that the expression of NMMHC-B would differ in balloon-dilated versus stented arteries. METHODS: To study the localisation and time course of expression of NMMHC-B, we performed stenting or balloon dilation in peripheral arteries of 16 atherosclerotic Yucatan micropigs and used serial intravascular ultrasound (IVUS) and angiography to measure geometric dimensions following balloon angioplasty or stenting. In situ hybridisation techniques were used to detect NMMHC-B mRNA. 5'-bromo-2'-deoxyuridine (BrdU) was administered to detect proliferating cells. By counting the number of silver grains in the different layers of the artery, we could compare the amount of expression at the different time points between the groups. RESULTS: In intima and media, NMMHC-B expression increased after balloon dilation and stenting and peaked at 7 days. In stented arteries, the expression of NMMHC-B remained high for up to 42 days after injury, whereas in balloon-dilated arteries it had normalised. In the adventitia of balloon-dilated arteries, but not of stented arteries, NMMHC-B expression peaked at 7 days. NMMHC-B expression was not limited to proliferating cells. CONCLUSION: NMMHC-B is expressed near sites of active repair after arterial injury, but not limited to proliferating cells. The different pattern of NMMHC-B expression after balloon dilation compared with stenting may be related to arterial remodelling, because stented arteries that do not remodel lack this conspicuous adventitial expression at 7 days.

The atherosclerotic Yucatan animal model to study the arterial response after balloon angioplasty: the natural history of remodeling.

OBJECTIVE: Remodeling in de novo atherosclerosis and in restenosis after balloon angioplasty constitutes a change in total arterial circumference which, together with plaque growth or neointimal formation, determines the lumen of the artery. To better understand the fundamental biology of neointimal formation, remodeling and their interaction, animal studies are needed. In this study, we described in detail the methodology used and the natural history of neointimal formation and remodeling after balloon angioplasty in atherosclerotic Yucatan micropigs. METHODS AND RESULTS: Atherosclerosis was induced in 60 peripheral arteries of sixteen Yucatan micropigs by a combination of denudation and atherogenic diet. Balloon angioplasty was performed in 38 arteries, with serial intravascular ultrasound (IVUS) and quantitative angiography before and after intervention and at 2, 4, 7, 14 or 42 days follow-up. Remodeling, expressed as late media-bounded area (MBA) loss, increased progressively over time. At 42 days, late MBA loss after balloon angioplasty was significantly different compared to late MBA loss in control arteries, 2.2 +/- 1.0 versus -0.3 +/- 1.1 mm2 and p = 0.02. Late lumen loss increased over time and was highest at 42 days after balloon angioplasty (2.8 +/- 0.7 mm2). The contribution of neointimal formation to late lumen loss decreased over time and the contribution of late MBA loss to late lumen increased over time and was highest at 42 days (78%). Medial necrosis was 48% at two days after balloon angioplasty and the repopulation of the media was almost completed at seven days. CONCLUSION: Remodeling following balloon angioplasty has an early onset and progresses with neointimal formation to cause restenosis over the standard 42-day time course for Yucatan micropigs. This correlates to six months renarrowing in humans. In this model, atherosclerosis and the natural history of restenosis, both with respect to neointimal formation and remodeling, resemble the human disease quite closely.

A novel one-shot anastomotic stapler prototype for coronary bypass grafting on the beating heart: feasibility in the pig.

OBJECTIVE: The nonpenetrating, arcuate-legged clip has proved its ability to provide a high-quality microvascular anastomosis. This study assessed the feasibility of constructing a coronary end-to-side anastomosis on the beating heart with a novel mechanical, sutureless anastomotic device that applies 12 circumferential clips simultaneously. METHODS: In 14 consecutive pigs (70-90 kg), the left internal thoracic artery (diameter, 3 mm) was grafted to the left anterior descending coronary artery (diameter, 3 mm) by means of a one-shot anastomotic stapler prototype. Endothelial denudation, medial necrosis, and intimal hyperplasia were analyzed quantitatively and compared with those seen in conventionally sutured anastomoses (n = 4). RESULTS: In 8 of 14 anastomoses, the one-shot anastomotic stapler successfully applied all 12 clips circumferentially across the everted arteriotomy edges. In the remaining, either 1 (n = 4) or 3 and 4 adjoining malaligned clips had to be replaced manually with a single-clip applicator. Coronary occlusion was limited to approximately 3 minutes. At follow-up, all anastomoses were patent angiographically. At 2 days, in 2 of 7 cases, a local coronary dissection was observed, and there was a considerable loss of endothelial cells and medial damage. At 28 days, however, minimal intimal hyperplasia was seen at the anastomotic lining, although more pronounced when compared with conventionally sutured anastomoses. CONCLUSIONS: The one-shot anastomotic stapler prototype enabled short-occlusive (3 minutes), sutureless end-to-side grafting on the beating porcine heart. In spite of early endothelial and medial damage and 2 local dissections, all anastomoses remained patent with minimal intimal hyperplasia at 4 weeks.

Arteriotomy closure by glued patch in the porcine carotid artery.

BACKGROUND: A thoracoscopic approach to coronary bypass grafting warrants renewed search for facilitated vascular anastomosis methods. We reassessed tissue adhesion, sealing properties, and histotoxicity of iso-butyl-cyanoacrylate, in a simplified anastomosis model. METHODS: In 12 Dutch landrace pigs, five arteriotomies were made in each carotid artery. The arteriotomies were closed by conventional microvascular suturing or by pericardial patch (Peri-Strips) and iso-butyl-cyanoacrylate. The animals were sacrificed at 2 hours (n = 2), 2 days (n = 5), and 4 weeks (n = 5). The arteries were examined by flow measurement, angiography, and histologic analysis. RESULTS: The time required to close arteriotomies by conventional suturing was 257 +/- 43 seconds (mean +/- SD) and by glued patch 51 +/- 27 seconds (p < 0.001). In all arteriotomies covered by glued patch, complete hemostasis was obtained. At 2 days and 4 weeks, with both methods the same histologic results were observed. At follow-up, all carotid arteries were patent without stenoses. CONCLUSIONS: Arteriotomy closure by glued patch was simple, fast, reliable, and without signs of histotoxicity. Adhesives deserve to be reconsidered as an alternative to suturing in closed chest beating-heart coronary surgery.

Temporary luminal arteriotomy seal: II. Coronary artery bypass grafting on the beating heart.

BACKGROUND: This study assessed the feasibility of applying a temporary luminal arteriotomy seal during end-to-side coronary artery bypass grafting on the beating heart. METHODS: In 18 consecutive pigs, the left internal mammary artery was grafted to the left anterior descending coronary artery, and the arteriotomy was temporarily sealed luminally by a 200-microm-thick polyurethane seal. Endothelial denudation, medial necrosis, and intimal hyperplasia were measured quantitatively and compared with conventionally sutured anastomoses (n=4 pigs). RESULTS: Insertion and retrieval of the seal required 28+/-12 and 11+/-6 seconds, respectively. Including the arteriotomy, coronary artery occlusion was limited to about 80 seconds. The seal provided a bloodless arteriotomy in all anastomoses with unimpeded coronary artery blood flow. Endothelial denudation was limited to two thirds of the circumference of the coronary artery. No medial necrosis was found. Intimal hyperplasia at the suture line was small, although more pronounced when compared with conventionally sutured anastomoses. CONCLUSIONS: In off-pump, beating-heart coronary artery bypass grafting, the temporary luminal arteriotomy seal provided a bloodless arteriotomy with negligible obstruction to coronary artery blood flow, and with a minimum of arterial wall damage. It is conceivable that this seal may expand the indications for coronary surgical procedures without cardiopulmonary bypass.

Temporary luminal arteriotomy seal: III. Postmortem arteriosclerotic human coronary artery.

BACKGROUND: Recently, we described a temporary luminal arteriotomy seal that provided a bloodless arteriotomy without obstructing recipient artery blood flow during bypass grafting in nonarteriosclerotic porcine arteries. This postmortem study assessed the sealing properties in irregular arteriosclerotic human coronary arteries. METHODS: Three hearts were obtained from donated corpses within 24 hours of death. The coronary arteries were pressure-perfused at 60 mm Hg with citrated porcine blood. At 15 anastomosis sites in four different coronary arteries, an end-to-side anastomosis was created using a 200-microm-thick polyurethane seal. Adequacy of sealing was determined at perfusion pressures of 60, 40, and 20 mm Hg. RESULTS: After insertion, the arteriotomy was sealed instantaneously in 10 of 15 anastomoses. After repositioning, complete sealing with a bloodless operative field was obtained in all cases. Low intracoronary transmural pressure did not impede sealing. In 8 of 15 anastomoses, minor leakage without obscuring the arteriotomy edges was observed during anastomotic suturing. Histologic examination revealed no intimal tear or dissection caused by the anastomotic procedure. CONCLUSIONS: In postmortem-obtained arteriosclerotic human coronary arteries, the temporary luminal arteriotomy seal provided optimal visualization of the coronary anastomosis site in combination with persistent distal perfusion.

Altered in vitro chondrogenic properties of chondrocytes harvested from unaffected cartilage in osteoarthritic joints.

OBJECTIVE: In vitro models of chondrogenesis often depart from chondrocytes harvested from less-affected areas of osteoarthritic joints. However, there are indications that these chondrocytes are phenotypically different from chondrocytes from healthy joints and thus might differ in their capacity to generate hyaline cartilage. The goal of this study was to compare the chondrogenic capacity of chondrocytes from healthy and OA joints. DESIGN: Chondrocytes isolated from nine healthy and nine OA knee joints were expanded in monolayer for two passages. Chondrocytes from passages 1 and 2 were analyzed for expression of (de)differentiation and hypertrophy markers and were seeded at passage 2 on collagen-coated filters for redifferentiation culture to study cartilage matrix formation. RESULTS: The collagen II/I mRNA ratio, reflecting differentiation, decreased from passage 1 to 2 in both chondrocytes from OA joints and chondrocytes from healthy joints (P<0.05), without a significant difference between the two donor types. At passage 1, levels of the cartilage transcription factors Sox-5, Sox-6 and Sox-9 appeared to be higher in chondrocytes from OA joints (n.s.), but this was not seen at passage 2. However, a clear difference was observed in collagen type X expression, which was high in chondrocytes from OA joints at both passages, while undetectable in chondrocytes from healthy joints (P<0.01). Tissue generated by chondrocytes from healthy joints redifferentiated for 28 days, showed a significantly better morphology, as assessed by histological scoring (P<0.01) and higher proteoglycan content (P<0.05), compared to chondrocytes from OA joints. Matrix turnover parameters, i.e., proteoglycan synthesis and degradation rate, were not significantly affected by donor tissue origin. CONCLUSIONS: These results suggest that clear differences between chondrocytes from healthy and OA joints exist and that these are not completely abolished during the process of de- and redifferentiation. Therefore, in vitro cartilage regeneration models, which use chondrocytes from OA joints, should be interpreted with care.

The relation between de novo atherosclerosis remodeling and angioplasty-induced remodeling in an atherosclerotic Yucatan micropig model.

Geometric remodeling in de novo atherosclerosis and in restenosis after balloon angioplasty constitutes a change in total arterial circumference that, together with plaque growth or neointima formation, determines the lumen of the artery. The heterogeneous nature of arterial obstructions raises the question of whether early and late outcomes (restenosis) of angioplasty are affected by the degree and direction of de novo atherosclerotic remodeling. This study was designed to assess the relationship between atherosclerotic remodeling and the degree and mechanism of restenosis after balloon angioplasty. Atherosclerosis was induced in 27 peripheral arteries of 18 Yucatan micropigs by a combination of denudation and atherogenic diet. Balloon angioplasty was performed, with serial intravascular ultrasound and quantitative angiography before and after intervention and at 42 days' follow-up. We used the relative media-bounded area (MBA), defined as the MBA of the treated site divided by the MBA of the reference, before angioplasty as a measure of remodeling in de novo atherosclerosis and late MBA loss as a measure of remodeling after balloon angioplasty. Relative MBA before angioplasty was not correlated with angiographic and echographic acute gain after balloon angioplasty (r=.22, P=.28 and r=.14, P=.48) or with late lumen loss (r=-.05, P=.81 and r=.19, P=.33). No correlation was found between relative MBA and late MBA loss (r=.14 and P=.48). In the atherosclerotic Yucatan micropig, remodeling during de novo atherosclerosis has no relevance for acute gain and late lumen loss after balloon angioplasty. Both the direction and the extent of remodeling after balloon angioplasty are not related to the direction and extent of remodeling during de novo atherosclerosis.

Relationship between plaque mass and neointimal hyperplasia after stent placement in Yucatan micropigs.

PURPOSE: To determine whether the amount of neointima found after stent placement is related to the preexistent plaque mass in the peripheral arteries in micropigs. MATERIALS AND METHODS: Twenty-six peripheral arteries were studied in 14 Yucatan micropigs in a denudation and diet-induced atherosclerosis model. Quantitative angiographic and intravascular ultrasound (US) analysis were performed before and after stent placement and at follow-up 6 weeks after stent placement. At follow-up, the peripheral arterial tree was fixed by means of pressure perfusion, processed for histologic analysis, and analyzed morphometrically with a computer-based system. RESULTS: Preexistent plaque correlated strongly with intimal hyperplasia measured on US images (r = .81, P < .001) or in histologic sections (r = .53, P = .006). No correlation was found between dilation ratio and intimal hyperplasia or between balloon-to-lesion ratio and intimal hyperplasia. In multivariate models of intimal hyperplasia, preexistent plaque and early gain in lumen diameter depicted at angiography were independent explanatory variables. CONCLUSION: The amount of plaque present before stent placement was a determinant of the amount of intimal hyperplasia present after stent placement.