Human umbilical cord cells: a new cell source for cardiovascular tissue engineering

BACKGROUND: Tissue engineering of viable, autologous cardiovascular constructs with the potential to grow, repair, and remodel represents a promising new concept for cardiac surgery, especially for pediatric patients. Currently, vascular myofibroblast cells (VC) represent an established cell source for cardiovascular tissue engineering. Cell isolation requires the invasive harvesting of venous or arterial vessel segments before scaffold seeding, a technique that may not be preferable, particularly in pediatric patients. In this study, we investigated the feasibility of using umbilical cord cells (UCC) as an alternative autologous cell source for cardiovascular tissue engineering. METHODS: Human UCC were isolated from umbilical cord segments and expanded in culture. The cells were sequentially seeded on bioabsorbable copolymer patches (n = 5) and grown in vitro in laminar flow for 14 days. The UCC were characterized by flow cytometry (FACS), histology, immunohistochemistry, and proliferation assays and were compared to saphenous vein-derived VC. Morphologic analysis of the UCC-seeded copolymer patches included histology and both transmission and scanning electron microscopy. Characterization of the extracellular matrix was performed by immunohistochemistry and quantitative extracellular matrix protein assays. The tissue-engineered UCC patches were biomechanically evaluated using uniaxial stress testing and were compared to native tissue. RESULTS: We found that isolated UCC show a fibroblast-like morphology and superior cell growth compared to VC. Phenotype analysis revealed positive signals for alpha-smooth muscle actin (ASMA), desmin, and vimentin. Histology and immunohistochemistry of seeded polymers showed layered tissue formation containing collagen I, III, and glycoaminoglycans. Transmission electron microscopy showed viable myofibroblasts and the deposition of collagen fibrils. A confluent tissue surface was observed during scanning electron microscopy. Glycoaminoglycan content did not reach values of native tissue, whereas cell content was increased. The biomechanical properties of the tissue-engineered constructs approached native tissue values. CONCLUSIONS: Tissue engineering of cardiovascular constructs using UCC is feasible in an in vitro environment. The UCC demonstrated excellent growth properties and tissue formation with mechanical properties approaching native tissue. It appears that UCC represent a promising alternative autologous cell source for cardiovascular tissue engineering, offering the additional benefits of using juvenile cells and avoiding the invasive harvesting of intact vascular structures.     

Human umbilical cord cells for cardiovascular tissue engineering: a comparative study.

OBJECTIVE: Tissue engineering of viable, autologous cardiovascular replacements with the potential to grow, repair and remodel represents an attractive approach to overcome the shortcomings of available replacements for the repair of congenital cardiac defects. Currently, vascular myofibroblast cells represent an established cell source for cardiovascular tissue engineering. Cell isolation requires the invasive harvesting of venous or arterial vessel segments prior to scaffold seeding, a technique which may not be preferable, especially in pediatric patients. This study evaluates cells isolated from human umbilical cord artery, umbilical cord vein and whole cord as alternative autologous cell sources for cardiovascular tissue engineering. METHODS: Cells were isolated from human umbilical cord artery (UCA), umbilical cord vein (UCV), whole umbilical cord (UCC) and saphenous vein segments (VC), and were expanded in culture. All three expanded cell groups were seeded on bioabsorbable copolymer strips and grown in vitro for 28 days. Isolated cells were characterized by flow cytometry, histology, immunohistochemistry, proliferation assays and compared to VC. Morphological analysis of the seeded polymer strips included histology, immunohistochemistry, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transmission electron microscopy (TEM), scanning electron microscopy (SEM) and uniaxial stress testing. RESULTS: UCA, UCV and UCC demonstrated excellent cell growth properties comparable to VC. Following isolation, all three cell groups showed myofibroblast-like morphology and characteristics by staining positive for alpha-smooth muscle actin (ASMA) and vimentin. Histology and immunohistochemistry of seeded polymers showed good tissue and extracellular matrix formation containing collagen I, III and elastin. TEM showed viable myofibroblasts and the deposition of collagen fibrils and progressive growing tissue formation, with a confluent surface, was observed in SEM. No difference was found among the mechanical properties of UCA, UCV, UCC and VC tissue engineered constructs. CONCLUSIONS: Tissue engineering of cardiovascular constructs by using UCA, UCV and UCC is feasible in an in vitro environment. Cell growth, morphology, characteristics and tissue formation were comparable between UCA, UCV, UCC and VC. UCC represent an attractive, readily available autologous cell source for cardiovascular tissue engineering offering the additional benefits of utilizing juvenile cells and avoiding the invasive harvesting of intact vascular structures.     

Living patches engineered from human umbilical cord derived fibroblasts and endothelial progenitor cells.

OBJECTIVE: A major shortcoming in contemporary congenital heart surgery is the lack of viable replacement materials with the capacity of growth and regeneration. Here we focused on living autologous patches engineered from human umbilical cord derived fibroblasts and endothelial progenitor cells (EPCs) as a ready-to-use cell source for paediatric cardiovascular tissue engineering. METHODS: EPCs were isolated from 20 ml fresh umbilical cord blood by density gradient centrifugation and myofibroblasts were harvested from umbilical cord tissue. Cells were differentiated and expanded in vitro using nutrient media containing growth factors. Before seeding, cell-phenotypes were assessed by immuno-histochemistry. Biodegradable patches fabricated from synthetic polymers (PGA/P4HB) were seeded with myofibroblasts followed by endothelialization with EPCs. All patches were cultured in a perfusion bioreactor. A subgroup of patches was additionally stimulated by cyclic strain. Analysis of the neo-tissues comprised histology, immuno-histochemistry, extracellular matrix (ECM) analysis and biomechanical testing. RESULTS: Endothelial phenotypes of EPCs before seeding were confirmed by Ac-Dil-LDL, CD 31, von-Willebrand-Factor and eNOS staining. Histology of the seeded patches demonstrated layered viable tissue formation in all samples. The cells in the newly formed tissues expressed myofibroblast markers, such as desmin and alpha-SMA. The EPCs derived neo-endothelia showed constant endothelial phenotypes (CD 31, vWF). major constituents of ECM such as collagen and proteoglycans were biochemically detected. Stress-strain properties of the patches showed features of native-analogous tissues. CONCLUSIONS: Living tissue engineered patches can be successfully generated from human umbilical cord derived myofibroblasts and EPCs. This new cell source may enable the tissue engineering of versatile, living, autologous replacement materials for congenital cardiac interventions.     

冷冻保存人脐带血管细胞培育组织工程心血管补片

目的 探讨应用液氮冷冻保存的人脐带动脉壁肌成纤维细胞(HUAMs)制备组织工程心血管补片的可行性.方法 将液氮冷冻保存的人脐带动脉壁肌成纤维细胞种植在聚-4-羟基丁酸酯(P4HB)聚合材料构建的补片支架材料上,制备组织工程心血管补片.体外静态培育21 d,对补片组织进行组织学、扫描电镜检查.免疫组织化学分析组织样本中细胞外基质(胶原蛋白)的合成.结果 苏木素-伊红(HE)染色显示,肌成纤维细胞较好地黏附于聚合材料上,大部分细胞保持生长活力,并浸润生长入支架材料内部,形成组织.免疫组织化学显示补片材料中有胶原蛋白合成.结论 液氮冷冻保存的人类脐带肌成纤维细胞可作为种子细胞,用于制备人组织工程心血管补片.     

脐静脉内皮细胞与双向羟基磷灰石联合培养的初步研究

目的:研究脐静脉内皮细胞在双向羟基磷灰石(BPM)的生长情况,为组织工程材料研究提供实验基础。方法:人脐静脉内皮细胞来源于永生细胞系,BPM紫外线照射30min ,70 0ml/L乙醇洗涤6 0min ,无菌滤纸吸干,用磷酸缓冲盐水洗涤3遍,每次30min ,而后用无菌滤纸吸干,将处理过的BPM材料泡入DMEM培养基(DMEM ) +2 0ml/L胎牛血清中过夜,使用前用无菌滤纸吸干。应用人脐静脉内皮细胞(HUVECs)和BPM进行混合培养,应用光学显微镜、荧光显微镜及扫描电子显微镜对其进行观察,并应用MTT测定细胞增殖情况。结果:人脐静脉内皮细胞在BPM表面生长、增殖良好,并可观察到细胞长入基质材料微孔内的改变。在培养的第3天,采用MTT法测定对照组与双向羟基磷灰石组的光吸收值,对照组为0 . 6 16±0. 0 17,双向羟基磷灰石组为0 . 6 38±0 . 0 18,两组比较无统计学差异(P >0 . 0 5 )。结论:双向羟基磷灰石(BPM )可作为细胞移植的载体。     

Tissue engineering in cardiovascular surgery: new approach to develop completely human autologous tissue.

OBJECTIVE: In cardiovascular tissue engineering, three-dimensional scaffolds serve as physical supports and templates for cell attachment and tissue development. Currently used scaffolds are still far from ideal, they are potentially immunogenic and they show toxic degradation and inflammatory reactions. The aim of this study is to develop a new method for a three-dimensional completely autologous human tissue without using any scaffold materials. METHODS: Human aortic tissue is harvested from the ascending aorta in the operation room and worked up to pure human myofibroblasts cultures. These human aortic myofibroblasts cultures (1.5x10(6) cells, passage 3) were seeded into 15-cm culture dishes. Cells were cultured with Dulbecco' s modified Eagle's medium supplemented with 1 mM L-ascorbic acid 2-phosphate for 4 weeks to form myofibroblast sheets. The harvested cell sheets were folded to form four-layer sheets. The folded sheets were then framed up and cultured for another 4 weeks. Tissue development was evaluated by biochemical assay and light and electron microscopy. RESULTS: After 4 weeks of culture in ascorbic acid supplemented medium, myofibroblasts formed thin cell sheets in culture dishes. The cell sheets presented in a multi-layered pattern surrounded by extracellular matrices. Cultured for additional 4 weeks on the frames, the folded sheets further developed into more solid and flexible tissues. Light microscopy documented a structure resembling to a native tissue with confluent extracellular matrix. Under transmission electron microscope, viable cells and confluent bundles of striated mature collagen fibers were observed. Hydroxyproline assays showed significant increase of collagen content after culturing on the frames and were 80.5% of that of natural human pericardium. CONCLUSIONS: Improved cell culture technique may render human aortic myofibroblasts to a native tissue-like structure. A three-dimensional completely autologous human tissue may be further developed on the base of this structure with no show toxic degradation or inflammatory reactions.     

胎儿附属物来源细胞的培养冻存

目的研究从胎儿附属物中分离培养及冻存细胞的较佳方法，以期为组织工程、细胞治疗和基因治疗提供种子细胞。方法取足月产胎儿脐带和胎盘，用酶消化法获得细胞，用相应培养液进行培养传代。将胎盘、脐带组织块冻存，检测加入不同浓度抗冻剂二甲亚砜的组织复苏后的活细胞率，透射电镜下观察其超微结构，并与新鲜组织进行比较；冻存培养所得人脐带血管周细胞和胎膜贴壁细胞的原代细胞，用免疫化学法检测冻存前后各自免疫表型的表达。结果新鲜脐带组织活细胞率为67．0％，抗冻剂体积分数为5％、10％、15％、20％时，冻存复苏脐带组织的活细胞率分别为23．4％、55．5％、48．8％、31．8％。抗冻剂体积分数为10％时，冻存复苏组织与新鲜组织活细胞率最接近（P〉0．05），体积分数为5％和20％时与新鲜组织该指标比较，差异均有统计学意义（P〈0．01）；透射电镜观察结果与之吻合。胎盘组织与脐带组织的情况类同。细胞冻存后免疫表型无明显变化。结论本方法可以从胎儿附属物中分离培养出大量种子细胞，冻存复苏后细胞免疫表型未发生改变，抗冻剂体积分数为10％时效果最好。     

Biocompatibility testing of different sterilised or disinfected allogenous bone grafts in comparison to the gold standard of autologous bone grafts--an "in vitro" analysis of immunomodulation

INTRODUCTION: Repair of large skeletal defects using bone allografts has become a routine procedure in orthopaedic and trauma surgery. Different procedures of sterilisation (82.5 degrees C disinfection; 121 degrees C autoclaving; PES; Tutoplast; 25 kGy gamma irradiation) are available to inactivate bacteria and fungi, including their spores, as well as viruses in human bone allografts. The efficiency of these procedures has been proven. However, the effects on the cellular response are rarely investigated. This present in vitro study investigates the immunological answer of human bone marrow cells to human allogenous and autologous bone platelets which were sterilised by different methods. MATERIALS AND METHODS: Human bone marrow cells and the bone platelets were harvested from patients undergoing a total hip replacement. All patients provided informed consent. Human bone platelets, 10 mm in diameter, 3 mm in height, were produced from femoral heads which were removed within the scope of total hip replacements. They were sterilised by different procedures or were disinfected (gamma radiotherapy, PES/ethanol treatment, Tutoplast procedure, 121 degrees C autoclaving, > 82.5 degrees C thermodisinfection). In addition, an autologous in vitro bone donation was simulated and compared with the allogenous bone grafts. Endobon was evaluated as a bovine hydroxyapatite ceramic. As control a human bone marrow cell culture without bone platelets was used. Over a period of four weeks the changes of the immunogenic cell populations were analysed in vitro (FACS analysis). Light and scanning microscopy were done to reveal morphological differences. As a vitality test the trypan-blue staining was performed. RESULTS: Light and scanning microscopy demonstrated large differences between the various sterilisation and disinfection methods. After 4 weeks the autologous bone platelets were completely covered with homogenously distributed human osteoblast like cells. The heat-sterilised/disinfected transplants demonstrated similar effects compared to the autologous bone grafts while the irradiated bone platelets demonstrated less cell coverage. 2/3 of the cells were vital on average after four weeks, with the exception of the irradiated bone platelets. The FACS analysis revealed in comparison to the control group provable differences in the immunological answer for the autologous bone donation as well as for the differently sterilised or disinfected allogenous bone grafts. The heat sterilisation or, respectively, disinfection methods compared to the autologous bone donation demonstrated almost similar in vitro effects. By far the worst results, characterised by an excessively increased portion of cytotoxic T-cells and a decreased amount of viable cells, were seen in the 25 kGy gamma irradiation samples. CONCLUSIONS: The results demonstrate the influence of the different sterilisation and disinfection procedures on the differentiation of human marrow cells (host). Similar in vitro effects were seen for the autologous and heat-treated bone platelets. The treatment of allogenous bone grafts with PES/ethanol and the Tutoplast procedures showed, just as Endobon, only low differences in comparison with the control cultures. The worse results in the case of the irradiated bone platelets may be explained by the production of free radicals which led to an excessive cell death.     

Electron microscopic observation of established chondrocytes derived from human intervertebral disc hernia (KTN-1) and role of macrophages in spontaneous regression of degenerated tissues.

BACKGROUND CONTEXT: Biological and pathological cell processes during the degeneration of intervertebral discs are as yet poorly understood. PURPOSE: An electron microscope was used to observe disc hernia degeneration at the cellular level as expressed in extruded tissue from a human intervertebral disc and in cultured chondrocytes. The mechanism of spontaneous regression was analyzed in order to investigate the effects of homologous macrophages, and the results of this analysis may be developed into a clinical therapy. STUDY DESIGN/SETTING: Extruded tissue specimens excised during surgery on human intervertebral disc hernia and cultured chondrocytes isolated from the excised tissue were observed by means of electron microscopy. Extracellular matrix metalloproteinase-3 (MMP-3) and its antagonist, tissue inhibitor of metalloproteinases-1 (TIMP-1), were observed by means of immune electron microscopy. Macrophages confirmed by CD68 immunostaining were added to the chondrocyte culture and observed by means of electron microscopy. PATIENT SAMPLE: All control subjects and patients gave written consent to the study. OUTCOME MEASURES: KTN-1 was directly observed without culture, and nuclei degeneration, the development of chromatin granules, changes in the osmotic pressure of the nuclear membrane and rough-surfaced endoplasmic reticulum, and the development of fat droplets were observed. METHODS: Tissues excised during surgery were divided, a part of the tissues were fixed in various fixatives for electron microscopy and immune electron microscopy analysis, and the other part was treated with collagenase. In addition, chondrocytes were isolated and cultured. Human peripheral blood mononuclear cells were separated using the Ficoll method. After culturing the cells, macrophages were collected, added to the chondrocyte culture, and observed under an electron microscope. CD68 positivity of the macrophages was confirmed by CD68 immunostaining. RESULTS: Freshly isolated chondrocytes in the hernia's extruded region differed markedly from cultured chondrocytes. By means of immunoelectron microscopy, MMP-3 and TIMP-1 were localized at the endoplasmic reticulum of the cultured chondrocytes. Infiltration of macrophages among the chondrocytes was observed in the mixed culture. CONCLUSIONS: The tissue extruded from the intervertebral disc showed obvious signs of degeneration, such as changes in osmotic pressure. Macrophages were observed to be the mechanism of spontaneous regression.     

脱细胞牛颈静脉血管支架的内皮化研究

目的 探讨在脱细胞牛颈静脉血管支架上进行内皮细胞再种植的可行性.方法 将人骨髓间充质干细胞(BMDCs)诱导分化为内皮种子细胞后种植在脱细胞牛颈静脉血管支架上,分为动态培养和静态培养2组.培养7 d后,对标本进行病理和扫描电镜观察.结果 静态培养的血管支架表面形成连续的单细胞层.动态培养后血管支架表面的细胞仍有50%残留,沿流场方向排列.结论 人骨髓间充质干细胞诱导分化的内皮种子细胞在脱细胞牛颈静脉血管支架上可以黏附生长.     

Tissue engineering of heart valves: in vitro experiences

BACKGROUND: Tissue engineering is a new approach, whereby techniques are being developed to transplant autologous cells onto biodegradable scaffolds to ultimately form new functional tissue in vitro and in vivo. Our laboratory has focused on the tissue engineering of heart valves, and we have fabricated a trileaflet heart valve scaffold from a biodegradable polymer, a polyhydroxyalkanoate. In this experiment we evaluated the suitability of this scaffold material as well as in vitro conditioning to create viable tissue for tissue engineering of a trileaflet heart valve. METHODS: We constructed a biodegradable and biocompatible trileaflet heart valve scaffold from a porous polyhydroxyalkanoate (Meatabolix Inc, Cambridge, MA). The scaffold consisted of a cylindrical stent (1 x 15 x 20 mm inner diameter) and leaflets (0.3 mm thick), which were attached to the stent by thermal processing techniques. The porous heart valve scaffold (pore size 100 to 240 microm) was seeded with vascular cells grown and expanded from an ovine carotid artery and placed into a pulsatile flow bioreactor for 1, 4, and 8 days. Analysis of the engineered tissue included biochemical examination, enviromental scanning electron microscopy, and histology. RESULTS: It was possible to create a trileaflet heart valve scaffold from polyhydroxyalkanoate, which opened and closed synchronously in a pulsatile flow bioreactor. The cells grew into the pores and formed a confluent layer after incubation and pulsatile flow exposure. The cells were mostly viable and formed connective tissue between the inside and the outside of the porous heart valve scaffold. Additionally, we demonstrated cell proliferation (DNA assay) and the capacity to generate collagen as measured by hydroxyproline assay and movat-stained glycosaminoglycans under in vitro pulsatile flow conditions. CONCLUSIONS: Polyhydroxyalkanoates can be used to fabricate a porous, biodegradable heart valve scaffold. The cells appear to be viable and extracellular matrix formation was induced after pulsatile flow exposure.     

In Vitro Generation of Atrioventricular Heart Valve Neoscaffolds

Tissue engineering of cardiovascular structures represents a novel approach to improve clinical strategies in heart valve disease treatment. The aim of this study was to engineer decellularized atrioventricular heart valve neoscaffolds with an intact ultrastructure and to reseed them with umbilical cord‐derived endothelial cells under physiological conditions in a bioreactor environment. Mitral (n = 38) and tricuspid (n = 36) valves were harvested from 40 hearts of German Landrace swine from a selected abattoir. Decellularization of atrioventricular heart valves was achieved by a detergent‐based cell extraction protocol. Evaluation of the decellularization method was conducted with light microscopy and quantitative analysis of collagen and elastin content. The presence of residual DNA within the decellularized atrioventricular heart valves was determined with spectrophotometric quantification. The described decellularization regime produced full removal of native cells while maintaining the mechanical stability and the quantitative composition of the atrioventricular heart valve neoscaffolds. The surface of the xenogeneic matrix could be successfully reseeded with in vitro‐expanded human umbilical cord‐derived endothelial cells under physiological flow conditions. After complete decellularization with the detergent‐based protocol described here, physiological reseeding of the xenogeneic neoscaffolds resulted in the formation of a confluent layer of human umbilical cord‐derived endothelial cells. These results warrant further research toward the generation of atrioventricular heart valve neoscaffolds on the basis of decellularized xenogeneic tissue.     

3D-Culturing of human osteoblastic cells with vessel like nutrient supply

AIM: The treatment of large, critical-size bone defects is a major therapeutic problem in orthopaedic and reconstructive surgery. The engineering of bone tissue could be used to replace lost bone mass. However, scaffolds seeded with vital cells and cultured in vitro suffer from poor oxygen and nutrient supply centrally, when the constructs exceed a critical volume. Therefore, we have established an osteoblastic cell culture in a new 3D-culture chamber with an artificial, vessel-like central membrane, allowing continuous nutrient supply. METHOD: Human osteoblasts were cultured in a 3D-like manner using a perfusion chamber for one week. In this system, the nutrient supply is guaranteed by a vessel-like, semipermeable polysulfone membrane with a continuous flow of medium. After fixation and cryosectioning, histological and immunohistological staining and scanning electron microscopy was carried out. RESULTS: Examinations reveal 3D cell growth around the central vessel. Formation of an extracellular matrix, rich in collagen type I and fibronectin, was detected immunohistochemically. Furthermore, we demonstrated cell adherence to the membrane and examined the surface morphology by scanning electron microscopy. CONCLUSION: The innovative approach for 3D-culturing of human osteoblasts in a system with a central nutrient supply opens up new possibilities for the in vitro cultivation for tissue engineering.     

人脐带间充质干细胞与蚕丝素多孔支架的体外复合培养

目的观察蚕丝素多孔支架对人脐带间充质干细胞（human umbilical cord mesenchymal stem cells,hUCMSCs）吸附作用及支架对hUCMSCs形态、功能及活性的影响,为脂肪组织工程支架选择提供实验依据。方法将hUCMSCs制成细胞悬液接种在蚕丝素多孔支架,荧光倒置相差显微镜、扫描电镜和四甲基偶氮唑蓝（MTT）法观察hUCMSCs的吸附和生长情况。结果培养1～2d后可见hUCMSCs与蚕丝素多孔支架充分附着。培养5～7d细胞生长增殖十分活跃,10d左右时,蚕丝素多孔支架孔中hUCMSCs成片状融合。荧光倒置相差显微镜和扫描电镜见细胞与支架黏附良好并有大量基质分泌,且活性指标与正常培养的hUCMSCs比较差异无统计学意义（P〉0．05）。结论蚕丝素多孔支架对hUCMSCs具有良好的吸附作用,并能维持其正常形态、功能及活性,蚕丝素多孔支架是hUCMSCs三维立体培养时的良好天然支架。     

Cultured human epithelium: human umbilical cord blood stem cells differentiate into keratinocytes under in vitro conditions

BACKGROUND: Stem cells have the capacity to renew or to give rise to a specialized cell types. Human umbilical cord blood (HUCB) has been explored as an alternative source of stem cells. However, its potential to differentiate into cells of other tissues is still under discussion. The aim of our study was to evaluate if HUCB stem cells could differentiate into epithelial cells under in vitro conditions. METHODS: Human keratinocytes derived from adult female skin donors, were isolated and cultured on fibrin glue/fibroblast gels-control group. In the umbilical cord blood cell group, male umbilical cord blood cells were added at a 1:10 ratio to keratinocytes and co-cultured on the fibrin glue/fibroblasts gel. After 15 days of culture, the sheets were analyzed by use of histochemistry and FISH. DNA was extracted and evaluated by use of polymerase chain reaction (PCR) for detection of Y-chromosome-specific sequences. RESULTS: In both groups a regular epithelial sheet consisting of three to four layers of cells was formed. Using PCR and FISH, in the umbilical cord blood cell group the presence of Y-chromosome-specific sequences in the cultured keratinocytes could be detected. In the control group, no Y-chromosome-specific sequences could be detected. CONCLUSION: Our findings indicate that umbilical cord blood stem cells differentiate into epithelial cells under in vitro conditions and thereby, might serve as a starting material for isolation and expansion of cells for transplantation in patients with large skin defects.     

A new source for cardiovascular tissue engineering: human bone marrow stromal cells

Objective: Vascular-derived cells represent an established cell source for tissue engineering of cardiovascular constructs. Previously, cell isolation was performed by harvesting of vascular structures prior to scaffold seeding. Marrow stromal cells (MSC) demonstrate the ability to differentiate into multiple mesenchymal cell lineages and would offer an alternative cell source for tissue engineering involving a less invasive harvesting technique. We studied the feasibility of using MSC as an alternative cell source for cardiovascular tissue engineering. Methods: Human MSC were isolated from bone marrow and expanded in culture. Subsequently MSC were seeded on bioabsorbable polymers and grown in vitro. Cultivated cells and seeded polymers were studied for cell characterization and tissue formation including extracellular matrix production. Applied methods comprised flow cytometry, histology, immunohistochemistry, transmission (TEM) and scanning electron microscopy (SEM), and biochemical assays. Results: Isolated MSC demonstrated fibroblast-like morphology. Phenotype analysis revealed positive signals for alpha-smooth muscle actin and vimentin. Histology and SEM of seeded polymers showed layered tissue formation. TEM demonstrated formation of extracellular matrix with deposition of collagen fibrils. Matrix protein analysis showed production of collagen I and III. In comparison to vascular-derived cell constructs quantitative analysis demonstrated comparable amounts of extracellular matrix proteins in the tissue engineered constructs. Conclusions: Isolated MSC demonstrated myofibroblast-like characteristics. Tissue formation on bioabsorbable scaffolds was feasible with extracellular matrix production comparable to vascular-cell derived tissue engineered constructs. It appears that MSC represent a promising cell source for cardiovascular tissue engineering.     

In vitro engineering of human autogenous cartilage.

A challenge in tissue engineering is the in vitro generation of human cartilage. To meet standards for in vitro-engineered cartilage, such as prevention of immune response and structural as well as functional integration to surrounding tissue, we established a three-dimensional cell culture system without adding exogenous growth factors or scaffolds. Human chondrocytes were cultured as spheroids. Tissue morphology and protein expression was analyzed using histological and immunohistochemical investigations on spheroid cryosections. A cartilage-like tissue similar to naturally occurring cartilage was generated when spheroids were cultured in medium supplemented only with human serum. This in vitro tissue was characterized by the synthesis of the hyaline-specific proteins collagen type II and S-100, as well as the synthesis of hyaline-specific mucopolysaccharides that increased with prolonged culture time. After 3 months, cell number in the interior of in vitro tissues was diminished and was only twice as much as in native cartilage. Additionally, spheroids quickly adhered to and migrated on glass slides and on human condyle cartilage. The addition of antibiotics to autologous spheroid cultures inhibited the synthesis of matrix proteins. Remarkably, replacing human serum by fetal calf serum resulted in the destruction of the inner part of the spheroids and only a viable rim of cells remained on the surface. These results show that the spheroid culture allows for the first time the autogenous in vitro engineering of human cartilage-like tissue where medium supplements were restricted to human serum.     

人脐带间充质干细胞向血管内皮祖细胞诱导分化的实验研究

目的探讨人脐带间充质干细胞定向诱导分化为血管内皮祖细胞的可行性,为构建组织工程血管瓣膜内皮化提供新的细胞来源。方法无菌条件下取剖宫产新生儿脐带,复合胶原酶消化法获取脐带间充质干细胞进行培养,以流式细胞仪检测脐带间充质细胞的表面标志。取扩增3～6代的脐带间充质干细胞用VEGF和b-FGF诱导分化。用免疫荧光法鉴定内皮祖细胞的标志。结果脐带间充质干细胞可以诱导分化为血管内皮样细胞,表达CD34、CD133和vWF,且Dil-acLDL实验阳性。结论脐带间充质干细胞可以诱导分化为血管内皮祖细胞,可为构建组织工程血管瓣膜提供新的种子细胞来源。     

种植人体活性细胞的生物心脏瓣膜

目前,种植人体活性细胞的生物心脏瓣膜主要有组织工程心脏瓣膜和种植人体活性细胞的猪主动脉瓣两种。组织工程心脏瓣膜是在人体可吸收的聚二醇酸纤维支架上种植人体同种活性细胞,先种植成纤维细胞,再种植单层内皮细胞包裹瓣叶。种植人体活性细胞的猪主动脉瓣是在清除原有细胞的组织内重建人体同种活性细胞。清除新鲜猪主动脉瓣呐原有细胞的方法是将瓣膜先经高、低渗溶液处理,然后用酶溶液处理。细胞经培养分离后,将成纤维细胞植入经处理的瓣膜组织,再植入内皮细胞。种植人体活性细胞的生物心脏瓣膜不会促使受者产生有害的免疫反应,并具有再生能力。     

Tissue engineering of vascular grafts: human cell seeding of decellularised porcine matrix.

OBJECTIVES: To develop a biocompatible and mechanically stable vascular graft combining human cells and a xenogenic acellular matrix. DESIGN/MATERIALS: Decellularised matrix tubes were obtained by enzymatic cell extraction of native porcine aortas. Endothelial cells and myofibroblasts were isolated from human saphenous veins and grown in cell cultures. The inner surface of the tubes was seeded with endothelial cells or myofibroblasts and exposed to pulsatile flow. RESULTS: After cell extraction, the absence of cellular components, as well as the maintenance of matrix integrity, was demonstrated by means of light microscopy and scanning electron microscopy. Furthermore, the porcine matrix was successfully seeded with human endothelial cells, which grew to a monolayer under flow conditions. Stable biomechanical properties were achieved at physiological perfusion pressures in vitro. CONCLUSIONS: Cellular components can be extracted from native porcine blood vessels. Vascular grafts can be generated in vitro of animal acellular matrix and human cells.