Raymond Labbé

In vitro construction of a human blood vessel from cultured vascular cells : a morphologic study

PURPOSE The purpose of this study was to create a tubular vascular model exclusively made of human cells and collagen. METHODS The blood vessel equivalent was constructed with the three following human cell types: vascular smooth muscle cells, endothelial cells, and fibroblasts. A tissuelike structure was obtained from the contraction of a tubular collagen gel (human origin) by vascular smooth muscle cells, which created a media-like structure. An adventitia-like tissue was added around the media-like structure by embedding fibroblasts into a collagen gel. An endothelium was established within the tubular structure after intraluminal cell seeding. RESULTS Cell orientation and gel contraction were followed up over time. Vascular smooth muscle cells developed a complex tridimensional network and were oriented in a circular fashion around the tubes axis. In contrast, fibroblasts were randomly oriented. A viable, homogeneous, and well-characterized endothelium was observed. These endothelial cells showed a slightly elongated structure and were oriented parallel to this vascular equivalent axis. CONCLUSION An in vitro tridimensional vascular model that exhibits some phenotypic characteristics of in vivo vascular cells could be useful in the study of events that lead to atherosclerotic plaque formations.

ISOLATION AND CULTURE OF THE THREE VASCULAR CELL TYPES FROM A SMALL VEIN BIOPSY SAMPLE

SummaryThe availability of small-diameter blood vessels remains a significant problem in vascular reconstruction. In small-diameter blood vessels, synthetic grafts resulted in low patency; the addition of endothelial cells (EC) has clearly improved this parameter, thereby proving the important contribution of the cellular component to the functionality of any construct. Because the optimal source of cells should be autologous, the adaptation of existing methods for the isolation of all the vascular cell types present in a single and small biopsy sample, thus reducing patient’s morbidity, is a first step toward future clinical applications of any newly developed tissue-engineered blood vessel. This study describes such a cell-harvesting procedure from vein biopsy samples of canine and human origin. For this purpose, we combined preexisting mechanical methods for the isolation of the three vascular cell types: EC by scraping of the endothelium using a scalpel blade, vascular smooth muscle cells (VSMC), and perivascular fibroblasts according to the explant method. Once in culture, cells rapidly grew with the high level of enrichment. The morphological, phenotypical, and functional expected criteria were maintained: EC formed cobblestone colonies, expressed the von Willebrand factor, and incorporated acetylated low-density lipoprotein (LDL); VSMC were elongated and contracted when challenged by vasoactive agents; perivascular fibroblasts formed a mechanically resistant structure. Thus, we demonstrated that an appropriate combination of preexisting harvesting methods is suitable to isolate simultaneously the vascular cell types present in a single biopsy sample. Their functional characteristics indicated that they were suitable for the cellularization of synthetic prosthesis or the reconstruction of functional multicellular autologous organs by tissue engineering.

Tissue-engineered vascular adventitia with vasa vasorum improves graft integration and vascularization through inosculation.

Tissue-engineered blood vessel is one of the most promising living substitutes for coronary and peripheral artery bypass graft surgery. However, one of the main limitations in tissue engineering is vascularization of the construct before implantation. Such a vascularization could play an important role in graft perfusion and host integration of tissue-engineered vascular adventitia. Using our self-assembly approach, we developed a method to vascularize tissue-engineered blood vessel constructs by coculturing endothelial cells in a fibroblast-laden tissue sheet. After subcutaneous implantation, enhancement of graft integration within the surrounding environment was noted after 48 h and an important improvement in blood circulation of the grafted tissue at 1 week postimplantation. The distinctive branching structure of end arteries characterizing the in vivo adventitial vasa vasorum has also been observed in long-term postimplantation follow-up. After a 90-day implantation period, hybrid vessels containing human and mouse endothelial cells were still perfused. Characterization of the mechanical properties of both control and vascularized adventitia demonstrated that the ultimate tensile strength, modulus, and failure strain were in the same order of magnitude of a pig coronary artery. The addition of a vasa vasorum to the tissue-engineered adventitia did not influence the burst pressure of these constructs. Hence, the present results indicate a promising answer to the many challenges associated with the in vitro vascularization and in vivo integration of many different tissue-engineered substitutes.

Erythromycin-Associated Ergotamine Intoxication: Arteriographic and Electrophysiologic Analysis of a Rare Cause of Severe Ischemia of the Lower Extremities and Associated Ischemic Neuropathy

Vasospasm associated with ergotamine is a well-known phenomenon. In this case report we present a rare drug interaction between erythromycin and ergotamine at normal doses causing lower extremity ischemia in a 36-year-old woman. Nitroprusside proved to be the treatment of choice. The response was dramatic and took place in a matter of hours in this patient. Ischemic monomelic neuropathy is a recently described entity in which axonal necrosis is caused by a loss of distal extremity blood flow. The association between erythromycin and ergotamine may be a dangerous pharmacologic combination; drugs that have a hepatic cycle with ergotamine derivatives must be used with caution.

Prospective study on the treatment of lower-extremity chronic venous and mixed ulcers using tissue-engineered skin substitute made by the self-assembly approach.

BACKGROUND: Despite present optimal standard treatment of lower-extremity ulceration, a high incidence of recurrence and treatment failure is observed. The objective of this project was to evaluate the effect of a self-assembled skin substitute (SASS) made by tissue engineering as a temporary cutaneous dressing in the treatment of hard-to-heal chronic ulcers. PATIENTS AND METHODS: The prospective uncontrolled case study includes patients suffering from venous or mixed ulcers lasting more than 6 months and unresponsive to compression therapy, with an Ankle Brachial Index greater than 0.5. Compression therapy was combined with the weekly application of SASS, produced from the patient’s own skin cells, until healing. A weekly follow-up recorded wound size, skin aspect, pain, drainage, and percentage of wound healing. Photographs were also taken to assess ulcer evolution. RESULTS: Fourteen ulcers present on 5 patients were treated. A mean of 6.7 SASS depositions by ulcer was required for healing. Two ulcers developed a minor wound infection, which was treated with oral antibiotics; another 2 ulcers recurred, and 1 healed with a second course of treatment, whereas 1 ulcer had a small recurrence treated with local wound care. CONCLUSION: The authors’ study suggests that the SASS used as a biological dressing is a promising treatment for hard-to-heal chronic venous and mixed ulcers that are unresponsive to compression therapy.

Fully thoracoscopic pulmonary lobectomy and specimen extraction through rib segment resection : Preliminary report

AbstractBackground: A technique of fully thoracoscopic pulmonary lobectomy with rib-segment resection for specimen extraction is described, and preliminary results in 18 patients are presented. Methods: Surgery is performed through four 15-mm ports. For all lobes except one, the surgeon operates in front of the patient, where the rib spaces are widest and rib-space trauma is less. When lobar dissection is complete, specimen extraction is performed after resection of a rib segment proportional to tumor size. Muscle section is kept to a minimum. There is no rib retraction. Results: There were no deaths, three conversions to open surgery, and three major complications. Average postoperative stay was 5.4 days for patients without complications and 9.6 days for patients with complications. In total six patients presented with some degree of air leaks, and two had post-thoracotomy pain (>2 months duration). The literature is reviewed to analyze current techniques and to define parameters of a truly minimally invasive pulmonary lobectomy. Conclusions: This technique is safe and promising; however, thoracoscopic lobectomy still needs refining. Before valid randomized studies comparing thoracoscopic lobectomy and muscle-sparing thoracotomy or posterolateral thoracotomy can be credible, technical issues related to the production of a truly minimally invasive procedure should be resolved.

Microstructured human fibroblast-derived extracellular matrix scaffold for vascular media fabrication.

In the clinical and pharmacological fields, there is a need for the production of tissue‐engineered small‐diameter blood vessels. We have demonstrated previously that the extracellular matrix (ECM) produced by fibroblasts can be used as a scaffold to support three‐dimensional (3D) growth of another cell type. Thus, a resistant tissue‐engineered vascular media can be produced when such scaffolds are used to culture smooth muscle cells (SMCs). The present study was designed to develop an anisotropic fibroblastic ECM sheet that could replicate the physiological architecture of blood vessels after being assembled into a small diameter vascular conduit. Anisotropic ECM scaffolds were produced using human dermal fibroblasts, grown on a microfabricated substrate with a specific topography, which led to cell alignment and unidirectional ECM assembly. Following their devitalization, the scaffolds were seeded with SMCs. These cells elongated and migrated in a single direction, following a specific angle relative to the direction of the aligned fibroblastic ECM. Their resultant ECM stained for collagen I and III and elastin, and the cells expressed SMC differentiation markers. Seven days after SMCs seeding, the sheets were rolled around a mandrel to form a tissue‐engineered vascular media. The resulting anisotropic ECM and cell alignment induced an increase in the mechanical strength and vascular reactivity in the circumferential direction as compared to unaligned constructs. Copyright