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[ε-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 × 10⁶ cells/cm² and 7 days of postseeding incubation were optimal for creating a confluent cellular layer on the polyglycolic acid/poly[ε-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.     

Small-diameter blood vessels engineered with bone marrow-derived cells

OBJECTIVE: The objective of this study is to investigate if bone marrow-derived cells (BMCs) regenerate vascular tissues and improve patency in tissue-engineered small-diameter (internal diameter = 3 mm) vascular grafts. SUMMARY BACKGROUND DATA: BMCs have demonstrated the ability to differentiate into endothelial-like cells and vascular smooth muscle-like cells and may offer an alternative cell source for vascular tissue engineering. Thus, we tissue-engineered small-diameter vascular grafts with BMCs and decellularized arteries. METHODS: Canine BMCs were differentiated in vitro into smooth muscle alpha-actin/smooth muscle myosin heavy-chain-positive cells and von Willebrand factor/CD31-positive cells and seeded onto decellularized canine carotid arteries (internal diameter = 3 mm). The seeded grafts were implanted in cell donor dogs. The vascular-tissue regeneration and graft patency were investigated with immunohistochemistry and angiography, respectively. RESULTS: The vascular grafts seeded with BMCs remained patent for up to 8 weeks in the canine carotid artery interposition model, whereas nonseeded grafts occluded within 2 weeks. Within 8 weeks after implantation, the vascular grafts showed regeneration of the 3 elements of artery (endothelium, media, and adventitia). BMCs labeled with a fluorescent dye prior to implantation were detected in the retrieved vascular grafts, indicating that the BMCs participated in the vascular tissue regeneration. CONCLUSIONS: Here we show that BMCs have the potential to regenerate vascular tissues and improve patency in tissue-engineered small-diameter vascular grafts. This is the first report of a small-diameter neovessel engineered with BMCs as a cell source.     

骨髓来源细胞构建组织工程血管补片

目的探讨组织工程方法构建血管补片的可行性。方法用壳聚糖和透明质酸多孔多聚体支架构建高分子复合材料支架，将犬的骨髓细胞种植其中，并植入自身犬的肺动脉上，观察材料上细胞外基质生成及表面内皮化的情况，分别在术后2周、4周、8周用电镜、组化等方法来进行评价。结果所有动物在实验期问均存活，组织学及免疫组织化学显示，术后补片形成类似血管壁样组织。结论此复合材料和骨髓细胞可用来构建组织工程血管补片，其结构与血管壁类似。     

Tissue engineering of heart valves in vivo using bone marrow-derived cells

In this study, we tissue-engineered heart valves in vivo using autologous bone marrow-derived cells (BMCs). Canine BMCs were differentiated into endothelial cell (EC)-like cells and myofibroblast (MF)-like cells. Decellularized porcine pulmonary valves were seeded with BMCs and implanted to abdominal aorta and pulmonary valve of bone marrow donor dogs. Histological examination of the explants identified the regeneration of valvular structures expressing CD31 and smooth muscle alpha-actin, indicating the presence of EC-like and MF-like cells in the grafts at 3 and 1 week, respectively, after implantation. Fluorescent microscopic examinations identified the presence of fluorescently labeled cells in the explants, indicating that the implanted BMCs survived and participated in the heart valve reconstitution. This study reports, for the first time, on tissue engineering of heart valve in vivo using BMCs.     

Midterm clinical result of tissue-engineered vascular autografts seeded with autologous bone marrow cells

OBJECTIVE: Prosthetic and bioprosthetic materials currently in use lack growth potential and therefore must be repeatedly replaced in pediatric patients as they grow. Tissue engineering is a new discipline that offers the potential for creating replacement structures from autologous cells and biodegradable polymer scaffolds. In May 2000, we initiated clinical application of tissue-engineered vascular grafts seeded with cultured cells. However, cell culturing is time-consuming, and xenoserum must be used. To overcome these disadvantages, we began to use bone marrow cells, readily available on the day of surgery, as a cell source. The aim of the study was to assess the safety and feasibility of this technique for creating vascular tissue under low-pressure systems such as pulmonary artery or venous pressure. METHODS: Since September 2001, tissue-engineered grafts seeded with autologous bone marrow cells have been implanted in 42 patients. The patients or their parents were fully informed and had given consent to the procedure. A 5-mL/kg specimen of bone marrow was aspirated with the patient under general anesthesia before the skin incision. The polymer tube serving as a scaffold for the cells was composed of a copolymer of l -lactide and -caprolactone (50:50). This copolymer is degraded by hydrolysis. The matrix is more than 80% porous, and the diameter of each pore is 20 to 100 microm. Polyglycolic acid woven fabric with a thickness of 0.5 mm was used for reinforcement. Twenty-three tissue-engineered conduits (grafts for extracardiac total cavopulmonary connection) and 19 tissue-engineered patches were used for the repair of congenital heart defects. The patients' ages ranged from 1 to 24 years (median 5.5 years). All patients underwent a catheterization study, computed tomographic scan, or both, for evaluation after the operation. The patients received anticoagulation therapy for 3 to 6 months after surgery. RESULTS: Mean follow-up after surgery was 490 +/- 276 days (1.3-31.6 months, median 16.7 months). There were no complications such as thrombosis, stenosis, or obstruction of the tissue-engineered autografts. One late death at 3 months after total cavopulmonary connection was noted in patient with hypoplastic left heart syndrome; this was unrelated to the tissue-engineered graft function. There was no evidence of aneurysm formation or calcification on cineangiography or computed tomography. All tube grafts were patent, and the diameter of the tube graft increased with time (110% +/- 7 % of the implanted size). CONCLUSION: Biodegradable conduits or patches seeded with autologous bone marrow cells showed normal function (good patency to a maximum follow-up of 32 months). As living tissues, these vascular structures may have the potential for growth, repair, and remodeling. The tissue-engineering approach may provide an important alternative to the use of prosthetic materials in the field of pediatric cardiovascular surgery. Longer follow-up is necessary to confirm the durability of this approach.     

Small intestinal submucosa as a small-caliber venous graft: a novel model for hepatocyte transplantation on synthetic biodegradable polymer scaffolds with direct access to the portal venous system

BACKGROUND/PURPOSE: Hepatotrophic factors in the portal blood are critically important for the survival of heterotopically transplanted hepatocytes. Currently, no model exists for the implantation of hepatocytes on biodegradable polymer scaffolds with direct access to the portal blood. This study investigates the use of small intestinal submucosa (SIS) as a small-caliber venous conduit that may be used for the implantation of tissue-engineered liver. METHODS: SIS was prepared from segments of rat jejunum and implanted as a venous conduit between the portal vein and inferior vena cava in 26 heparinized Lewis rats. Venograms were performed periodically, and the grafts were harvested at various time-points and examined by scanning electron microscopy (SEM) and histology. Von Willebrand Factor (vWF) staining was performed to assess endothelialization. RESULTS: Five rats died of technical complications. Seventeen of 21 rats (81%) maintained patent grafts at the time of death up to 8 weeks. Venograms demonstrated patent grafts at 3 and 8 weeks. SEM results showed a smooth luminal surface with endothelial-like cells by 3 weeks. Histology demonstrated a confluent luminal endothelial monolayer, absence of thrombus, and neovascularization in the SIS graft. VWF staining results were positive, confirming the growth of endothelial cells on the luminal surface. In preliminary studies, implantation of hepatocytes seeded on biodegradable polymer tubes into the SIS graft demonstrated clusters of viable cells after 2 days. CONCLUSIONS: Rat SIS can be prepared readily, maintains high patency as a small-caliber venous graft, and may be a useful model for the transplantation of tissue-engineered liver with access to the portal circulation.     

组织工程人工血管支架的预构

目的：探讨具有血管平滑肌细胞（vascular smooth muscle cells,VSMCs）活性的组织工程人工血管支架的构建方法。方法：①将聚羟基乙酸（polyglycolic acid,PGA）纤维无纺网支架材料、血管平滑肌细胞和胶原纤维相混合，构建组织工程血管。②将VSMCs置入胶原凝胶中，观察VSMCs的生长状况。③观察VSMCs胶原悬液滴入该支架材料中的生长。结果：①VSMCs在胶原凝胶中的位置固定，分布于不同层次，约3－4h逐渐形成多个胞质突起。随时间推移，胞质突起继续伸展，部分细胞呈梭形、纺锤形。②VSMCs胶原悬液滴入胶原包埋处理的PGA支架中后，大部分细胞随胶原凝胶状态的形成，滞留于支架材料网孔中，细胞可沿PGA纤维表面生长。结论：胶原包埋处理的PGA纤维无纺网是良好的携带VSMCs的多孔生物降解材料。     

Clinical results of tissue-engineered vascular autografts seeded with autologous bone marrow cells

In May 2000 we initiated clinical application of tissue-engineered (TE) vascular grafts seeded with cultured cells. However, cell culturing is time-consuming and xeno-serum must be used. To overcome these disadvantages, we started to use bone marrow cells (BMCs), readily available on the day of surgery, as a cell source. The aim of this study was to assess the safety and feasibility of this technique for creating pulmonary arteries. METHODS: Since August 2000, TE grafts seeded with autologous BMCs have been implanted in 42 patients. Bone marrow (5 ml/kg) was aspirated under general anesthesia prior to the skin incision. The polymer tube serving as a scaffold for the cells was composed of a co-polymer of 1-lactide and epsilon-caprolactone (50:50). This co-polymer is degraded by hydrolysis. The matrix is >80% porous and the diameter of each pore is 100-200 microm. Polyglycolic acid woven fabric with a thickness of 0.5 mm was used for reinforcement. Twenty-three TE conduits (TCPC grafts) and 19 TE patches were used for the repair of congenital heart defects. The patients' ages ranged from 1 to 24 years (median, 5.5 years). All patients underwent a catheterization study and/or computed tomography (CT) scans for evaluation after surgery. The patients received anticoagulation therapy for 3 to 6 months after surgery. RESULTS: Mean follow-up after surgery was 584 days (maximum 42 months). There were no lethal complications such as thrombosis or aneurysmal rupture. One late death at 3 months after TCPC was noted in a hypoplastic left heart syndrome patient, which was unrelated to the TE graft. There was no evidence of aneurysm formation on cineangiography or CT. CONCLUSIONS: Biodegradable conduits or pulmonary vessel patches seeded with autologous BMCs showed normal function (good patency up to maximum follow-up of 38 months). As living tissues, these vessels may have the potential for growth, repair, and remodeling. The TE approach may provide an important alternative to the use of prosthetic materials in the field of pediatric cardiovascular surgery. Longer follow-up is necessary to confirm the feasibility of this approach.     

Advances in tissue engineering of blood vessels and other tissues

Tissue engineering is a new and rapidly expanding field, in which techniques are being developed for culturing a variety of tissues both in vitro and in vivo using polymer ‘scaffolds’ to support tissue growth. Polymer scaffolds used in tissue engineering are generally biodegradable, often involving compounds which are already approved for human implantation. In some cases, these polymers may be chemically modified to exhibit selective cell adhesion properties, which enhance cell attachment and subsequent tissue growth. Many cell types have been successfully cultured on these scaffolds, including smooth muscle cells, endothelial cells, hepatocytes and chondrocytes.Tissue engineering holds the potential for the in vitro development of autologous or allogeneic transplantable vascular conduits. Each year in the USA, there are approximately 1.4 million procedures performed which require arterial prostheses. Most of these procedures are in small calibre (<6 mm) vessels, for which synthetic graft materials are not generally suitable. While autologous venouos or arterial vessels are generally used, not all patients possess adequate conduit for revascularization.Tubular scaffolds have been specially designed for culturing small calibre arteries in vitro. Bovine aortic vascular cells were seeded and cultured on these polymer scaffolds, and grown under conditions of pulsatile pressure and intra-luminal flow. To minimize contamination during the weeks of tissue culture required to produce an arterial prosthesis, a sterile incubator system was developed. Preliminary studies have achieved good cell densities of both smooth muscle cells and endothelial cells on biodegradable polymer scaffolds.     

Assessment of a tissue-engineered gastric wall patch in a rat model

Stenosis or deformity of the remaining stomach can occur after gastrectomy and result in stomach malfunction. The objective of this study is to demonstrate the feasibility of transplanting a tissue-engineered gastric wall patch in a rat model to alleviate the complications after resection of a large area of the gastric wall. Tissue-engineered gastric wall patches were created from gastric epithelial organoid units and biodegradable polymer scaffolds. In the first treatment group, gastric wall defects were created in recipient rats and covered with fresh tissue-engineered gastric wall patches (simultaneous transplantation). In the second treatment group, the tissue-engineered gastric wall patches were frozen for 12weeks, and then transplanted in recipient rats (metachronous transplantation). Tissue-engineered gastric wall patches were successfully used as a substitute of the resected native gastric wall in both simultaneous and metachronous transplantation groups. The defrosted wall patches showed almost the same cell viability as the fresh ones. Twenty-four weeks after transplantation, the defect in the gastric wall was well-covered with tissue-engineered gastric wall patch, and the repaired stomach showed no deformity macroscopically in both groups. Histology showed continuous mucosa and smooth muscle layers at the tissue-engineered stomach wall margin. The feasibility of transplanting a tissue-engineered patch to repair a defect in the native gastric wall has been successfully shown in a rat model, thereby taking one step closer toward the transplantation of an entire tissue-engineered stomach in the future.     

Tissue-engineered urinary bladder wall using PLGA mesh-collagen hybrid scaffolds: a comparison study of collagen sponge and gel as a scaffold

Purpose: Tissue engineering of the urinary bladder using autologous cells and biodegradable scaffold is a promising method for augmentation. The authors developed 2 hybrid scaffolds by combining poly (DL-lactic-co-glycolic acid; PLGA) mesh for mechanical strength with collagen sponge or gel suitable for cell seeding. The aim of this study was to compare collagen as a scaffold between collagen sponge and gel and to construct a tissue-engineered urinary bladder wall utilizing these hybrid scaffolds.Methods: The PLGA mesh-collagen hybrid scaffolds were prepared by introducing collagen sponge or gel into the PLGA knitted mesh. Urothelial and smooth muscle cells were obtained from porcine urinary bladder wall and were cultured in their respective media. The cells were seeded on these hybrid scaffolds. These constructs were analyzed morphologically and immunohistochemically.Results: The urothelial layer was generated 3 dimensionally by culturing urothelial cells with PLGA mesh and collagen sponge. The smooth muscle layer was constructed by culturing smooth muscle cells with PLGA mesh and collagen gel. And a novel tissue-engineered urinary bladder wall was constructed laminating the urothelial and smooth muscle layers.Conclusions: Ex vivo construction of urinary bladder wall using hybrid scaffolds prepared by combining PLGA mesh with collagen sponge or gel was successful. This tissue-engineered urinary bladder wall allows easy handling and may become a promising tool for bladder augmentation.     

The induction of angiogenesis by the implantation of autologous bone marrow cells: a novel and simple therapeutic method.

BACKGROUND: Bone marrow contains many kinds of primitive cells that could differentiate to endothelial cells and secrete several growth factors. In the current study, we attempted to induce therapeutic angiogenesis by implanting autologous bone marrow cells (BMCs) and using a rat ischemic hind limb model. METHODS: BMCs were prepared by removing red blood cells. A rat ischemic hind limb model was made by the ligation of the left femoral artery and its branches. BMCs were injected into 7 points of the ischemic muscles. To assess angiogenesis, a microangiogram, laser Doppler, and histologic evaluation were performed after the surgical procedure. RESULTS: A microangiogram and histologic evaluation showed that angiogenesis was significantly induced in the ischemic hind limb by the implantation of BMCs. Laser Doppler imaging analysis showed that blood flow was significantly increased after implantation of BMCs. Some implanted BMCs were stained positively with CD31 and vascular endothelial-cadherin (VE-cadherin), which might have been incorporated into the vasculature. The condition of ischemia caused an elevation in the level of basic fibroblast growth factor in the ischemic muscle and also in interleukin-1beta derived from the implanted BMCs, which might contribute to angiogenesis. CONCLUSION: These findings indicate that autologous bone marrow implantation may be a novel and simple method for inducing therapeutic angiogenesis.     

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 ?-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.     

生物可降解材料聚乳酸/聚羟基乙酸复合壳聚糖在人工冠状动脉血管支架制备中的应用

第一代金属裸支架和第二代涂层支架介入治疗冠状动脉粥样硬化性心脏病(冠心病)已得到广泛应用。由于长期存在金属支架异物刺激及其携带的药物扰乱血管壁各层细胞生长,引起支架内再狭窄和血管栓塞,远期仍有较多的主要心血管不良事件发生和需要再血管化治疗。因此,由聚酯、聚碳酸酐及聚磷酸酯等高分子材料制备的完全可生物降解吸收支架及药物洗脱支架应运而生,其中聚乳酸(poly-lactic acid,PLA)、聚羟基乙酸(poly-glycolicacid,PGA)、壳聚糖、聚己内酯(poly-caprolactone,PCL)及一些共聚物如聚乳酸/聚羟基乙酸共聚物(poly-lactic-co-glycolic acid,PLGA)材料制备的心血管植入支架的安全性、组织及血液相容性已得到证实,然而这些支架具有各自的缺点,如PLA降解较慢质硬易断裂柔韧性不足,PGA降解较快质软支撑力不足,支架降解太快或者太慢,均难以达到有效支撑,支架植入后容易出现血管损伤、弹性回缩,导致血管再狭窄及血栓形成,远期效果不佳。通过优化组合不同摩尔比的PLA和PGA及壳聚糖涂层,可以获得具有更好的生物相容性、适度的降解速率(约3～6个月完全降解)、足够的机械强度、较低的炎症反应和伸展度良好的复合材料,从而为制备完全生物可降解冠状动脉支架奠定实验基础。     

Tissue-engineered vascular grafts: does cell seeding matter?

Purpose

Use of tissue-engineered vascular grafts (TEVGs) in the repair of congenital heart defects provides growth and remodeling potential. Little is known about the mechanisms involved in neovessel formation. We sought to define the role of seeded monocytes derived from bone marrow mononuclear cells (BM-MNCs) on neovessel formation.

Methods

Small diameter biodegradable tubular scaffolds were constructed. Scaffolds were seeded with the entire population of BM-MNC (n = 15), BM-MNC excluding monocytes (n = 15), or only monocytes (n = 15) and implanted as infrarenal inferior vena cava (IVC) interposition grafts into severe combined immunodeficiency/bg mice. Grafts were evaluated at 1 week, 10 weeks, or 6 months via ultrasonography and microcomputed tomography, as well as by histologic and immunohistochemical techniques.

Results

All grafts remained patent without stenosis or aneurysm formation. Neovessels contained a luminal endothelial lining surrounded by concentric smooth muscle cell layer and collagen similar to that seen in the native mouse IVC. Graft diameters differed significantly between those scaffolds seeded with only monocytes (1.022 ± 0.155 mm) and those seeded without monocytes (0.771 ± 0.121 mm; P = .021) at 6 months.

Conclusions

Monocytes may play a role in maintaining graft patency. Incorporation of such findings into the development of second-generation TEVGs will promote graft patency and success.     

Undifferentiated mesenchymal stem cells seeded on a vascular prosthesis contribute to the restoration of a physiologic vascular wall

BACKGROUND: We evaluated the possibility of restoring a physiologic vascular wall using undifferentiated mesenchymal stem cells (MSCs) seeded on a polyurethane vascular prosthesis. METHODS: Undifferentiated MSCs were seeded on a vascular prosthesis and implanted into Wistar male rats (weight, 350 g) to investigate differentiation into smooth muscle cells and to determine graft endothelialization in vivo. RESULTS: Seeded or nonseeded grafts were surgically implanted. Undifferentiated MSCs were first labelled for green fluorescent protein. After 2 weeks in vivo, MSC that were initially self-expanded on the graft in a monolayer were organized in a multicellular layer mimicking media of aortic adjacent wall. They coexpressed green fluorescent protein and smooth muscle proteins that were not present before the in vivo engraftment, indicating that in vivo conditions induced smooth muscle protein maturation. Undifferentiated MSC showed an electrophysiologic profile quite different than mature smooth muscle cells. In both in vitro- and in vivo-differentiated MSCs, adenosine triphosphate, an IP(3)-dependent agonist, induced an increase in calcium similar to that which occurred in mature smooth muscle cells. However, MSCs failed to respond to caffeine, a ryanodine receptor activator, indicating the absence of mature calcium signaling, and finally, contraction was absent. Endothelialization attested by immunohistology and scanning electron microscopy was greater in MSC-seeded grafts that prevent thrombosis. CONCLUSION: Only partial smooth muscle cell differentiation of MSCs resulted when seeded on vascular grafts, but MSCs spontaneously restore a media-like thick wall. Mesenchymal stem cells have a positive impact on in vivo endothelialization in rats that supports their potential for use in vascular surgery. CLINICAL RELEVANCE: Thrombosis of vascular prostheses is a major complication of surgery. We showed on rat aorta that mesenchymal stem cells seeded on polyurethane patch restore endothelium. It also induced incomplete smooth muscle differentiation. In the future, stem cell could prevent thrombosis of vascular prostheses.     

Accelerated migration and proliferation of smooth muscle cells cultured from neointima induced by a vena cava filter

Formation of a neointima is associated with grafted artery or vein, angioplasty, and stent and inferior vena cava filter (IVCF) implantation. Contributing to the neointima is a population of vascular smooth muscle cells (SMC) that migrates from media and subsequently proliferates within intima. The purpose of this present study was to culture SMC from normal vessel wall and from neointima and to compare migration and growth of these cells. Neointima was stimulated in the vena cava of pigs by placement of an IVCF for 30 days. Tissue was taken from the thickened wall between the struts and from a normal segment of the IVCF. After removal of the endothelium and adventitia, explants were placed in culture dishes and were observed for the migration of cells. Immunoassay for smooth muscle alpha-actin was used to identify cell origin. Proliferation was determined by cell counting. The cell cycle regulator cyclin D1 was detected by Western blot analysis. SMC phenotype was confirmed by positive immunostaining for smooth muscle alpha-actin. Cells migrated from the neointimal explants (NI-SMC) more rapidly than cells from explants of normal media (NM-SMC). Proliferation of NI-SMC was also more rapid than NM-SMC with or without exogenous mitogens. NI-SMC expressed more cyclin D1 than NM-SMC. Injury to the vena cava triggered neointima formation characterized by the expansion of a population of SMC with increased migration and replication compared with SMC from normal regions of the vessel.     

Tissue engineering for cartilage repair: in vitro properties of a hyaluronan-derivative

Association of biomaterials with autologous cells can provide a new generation of implantable devices for cartilage and bone repair. Such scaffolds should provide a performed three-dimensional shape, prevent cells from floating out of the defect, have sufficient mechanical strength, facilitate uniform spread of cells, and stimulate the phenotype of transplanted cells. Hyaff-11 is a recently developed hyaluronic-acid based biodegradable polymer, that has been shown to provide successful cell scaffolds for tissue-engineered repair. The aim of this study was to evaluate in vitro the potential of Hyaff-11 to support the growth of human chondrocytes and to maintain their original phenotype. Our data indicate that human chondrocytes seeded on Hyaff-11 express and produce collagen type II and aggrecan and downregulate the production of collagen type I. These results provide an in vitro demonstration of therapeutic potential of Hyaff-11 as a delivery vehicle in tissue-engineered repair of articular cartilage defects.     

基于内皮祖细胞构建的组织工程静脉体内初步评价

目的探讨基于犬骨髓源内皮祖细胞和猪脱细胞动脉基质构建的组织工程静脉植入体内的可行性。方法实验犬（n=8）骨髓体外扩增获取内皮祖细胞,荧光标记后将其种植于猪脱细胞动脉基质,体外构建组织工程静脉。以未种植细胞的脱细胞基质为对照（n=4）,将其置换自体犬下腔静脉,术后10d、4周、12周造影后取材检测。结果实验（对照）组在第10天、4周、12周的通畅率分别为7／7（2／4）、6／6（2／2）和4／4（1／2）。相关检测提示实验组移植血管腔面覆盖汇合的内皮细胞,部分为种植的细胞,部分为新形成的内皮细胞,中层有成纤维细胞和α-actin阳性细胞出现。通畅的对照组腔面由假内膜覆盖。结论基于自体内皮祖细胞和脱细胞动脉基质构建组织工程静脉是可行的,但需要进一步改进和观察。     

Bladder autoaugmentation using various biodegradable scaffolds seeded with autologous smooth muscle cells in a rabbit model

Background/Purpose

The prolapsed mucosa after bladder autoaugmentation usually collapses, and the volume increment is limited. This study is aimed at evaluating the efficacy of autoaugmentation assisted with 2 different scaffolds, polyglycolic acid (PGA) mesh and small intestinal submucosa (SIS), seeded with autologous bladder smooth muscle cells in a rabbit model.

Methods

One month after an initial 70% partial cystectomy, various autoaugmentation surgeries were performed. These procedures included traditional autoaugmentation (n = 6) and traditional autoaugmentation covered with PGA or SIS without cell seeding (N) (PGA-N, n = 6; SIS-N, n = 6) or covered with scaffolds seeded with autologous bladder smooth muscle cells (C) (PGA-C, n = 6; SIS-C, n = 6). All were followed up by bladder volume measurement and retrieved on 1, 2, 3, and 6 months. Statistical analysis was by analysis of variance.

Results

A normal urothelial layer was maintained in all groups. Only PGA-C group showed a significant bladder capacity increment as compared with the other groups in all time-points (P = .001, .000, .000, and .001 at first, second, third, and sixth months, respectively). The PGA-C group showed grossly normal bladder wall with scattered smooth muscle bundles. The other groups had marked graft shrinkage with only unorganized muscle fibers.

Conclusion

Cell-seeded PGA polymer facilitates smooth muscle regeneration, offers sufficient bladder wall backup, and achieves satisfactory volume increment after the autoaugmentation with time. The collagen matrix, although seeded with cells, did not offer adequate mechanical support after the surgery.