Bone marrow as a cell source for tissue engineering heart valves

Background

This study was designed to assess the feasibility of using ovine bone marrow-derived mesenchymal stem cells to develop a trileaflet heart valve using a tissue engineering approach.

Methods

Bone marrow was aspirated from the sternum of adult sheep. Cells were isolated using a Ficoll gradient, cultured, and characterized based on immunofluorescent staining and the ability to differentiate down a specific cell lineage. Two million cells per centimeter squared were delivered onto a polyglycolic acid (PGA), poly-4-hydroxybutyrate (P4HB) composite scaffold and cultured for 1 week before being transferred to a pulse duplicator for an additional 2 weeks. The tissue-engineered valves were assessed by histology, scanning electron microscopy, and biomechanical flexure testing.

Results

Cells expressed SH2, a marker for mesenchymal stem cells, as well as specific markers of smooth muscle cell lineage including α-smooth muscle actin, desmin, and calponin. These cells could be induced to differentiate down an adipocyte lineage confirming they had not fully committed to a specific cell lineage. Preliminary histologic examination showed patchy surface confluency confirmed by scanning electron microscopy, and deep cellular material. Biomechanical flexure testing of the leaflets showed an effective stiffness comparable to normal valve leaflets.

Conclusions

Mesenchymal stem cells can be isolated noninvasively from the sternum of sheep and can adhere to and populate a PGA/P4HB composite scaffold to form “tissue” that has biomechanical properties similar to native heart valve leaflets. Thus, bone marrow may be a potential source of cells for tissue engineering trileaflet heart valves, particularly in children with congenital heart disease.     

Selection of cell source for ligament tissue engineering

Use of appropriate types of cells could potentially improve the functionality and structure of tissue engineered constructs, but little is known about the optimal cell source for ligament tissue engineering. The object of this study was to determine the optimal cell source for anterior cruciate ligament (ACL) tissue engineering. Fibroblasts isolated from anterior cruciate ligament, medial collateral ligament (MCL), as well as bone marrow mesenchymal stem cells (MSC) were compared using the following parameters: proliferation rate, collagen excretion, expression of collagen type I, II, and III, as well as alpha-smooth muscle actin. Green fluorescent protein (GFP) transfected MSCs were used to trace their fate in the knee joints. MSC, ACL, and MCL fibroblasts were all highly stained with antibodies for collagen types I and III and alpha-smooth muscle actin while negatively stained with collagen type II. Proliferation rate and collagen excretion of MSCs were higher than ACL and MCL fibroblasts (p < 0.05), and MSCs could survive for at least 6 weeks in knee joints. In summary, MSC is potentially a better cell source than ACL and MCL fibroblasts for anterior cruciate ligament tissue engineering.     

Thoracic Surgery Directors Association Award. Bone marrow as a cell source for tissue engineering heart valves

BACKGROUND: This study was designed to assess the feasibility of using ovine bone marrow-derived mesenchymal stem cells to develop a trileaflet heart valve using a tissue engineering approach. METHODS: Bone marrow was aspirated from the sternum of adult sheep. Cells were isolated using a Ficoll gradient, cultured, and characterized based on immunofluorescent staining and the ability to differentiate down a specific cell lineage. Two million cells per centimeter squared were delivered onto a polyglycolic acid (PGA), poly-4-hydroxybutyrate (P4HB) composite scaffold and cultured for 1 week before being transferred to a pulse duplicator for an additional 2 weeks. The tissue-engineered valves were assessed by histology, scanning electron microscopy, and biomechanical flexure testing. RESULTS: Cells expressed SH2, a marker for mesenchymal stem cells, as well as specific markers of smooth muscle cell lineage including alpha-smooth muscle actin, desmin, and calponin. These cells could be induced to differentiate down an adipocyte lineage confirming they had not fully committed to a specific cell lineage. Preliminary histologic examination showed patchy surface confluency confirmed by scanning electron microscopy, and deep cellular material. Biomechanical flexure testing of the leaflets showed an effective stiffness comparable to normal valve leaflets. CONCLUSIONS: Mesenchymal stem cells can be isolated noninvasively from the sternum of sheep and can adhere to and populate a PGA/P4HB composite scaffold to form "tissue" that has biomechanical properties similar to native heart valve leaflets. Thus, bone marrow may be a potential source of cells for tissue engineering trileaflet heart valves, particularly in children with congenital heart disease.     

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.     

血管组织工程中内皮种子细胞的干细胞来源研究进展

自体动脉或静脉是冠脉和外周旁路分流术中最常用的血管替代物,但由于损伤、血管疾病、先前的手术等原因,许多患者的自体血管并不合适。早期血管替代物的研究主要集中在膨化聚四氟乙烯、涤纶等人工合成材料构建的血管,但是,单纯由人工合成多聚物构建的血管常常由于早期的血栓形成导致移植失败,尤其对于小口径（小于6mm）或低血流量血管。     

A honeycomb collagen carrier for cell culture as a tissue engineering scaffold

As a three-dimensional carrier for cell culture, a honeycomb structure cell scaffold was created from atelopeptide collagen Types I, II, and III. The diameter of the honeycomb pores ranged from 100 to 1,000 microm. The depth of the pores was from 10 to 3,000 mm. The scaffold was elastic and hard. Creation of various shapes was easy, and these shapes were easily maintained. Human fibroblasts, CHO-K1, BHK-21, and bovine endothelial cells were cultured with the scaffold. The growth curves of these cells were satisfactory. These results suggest that this carrier is a suitable scaffold for cell culture and will be useful as a three-dimensional tissue engineering scaffold.     

Xenotransplantation of fetal porcine hepatocytes in rats using a tissue engineering approach

Hepatocytes can be successfully transplanted into highly vascular sites such as the spleen, liver, and lungs. Subcutaneous sites lack adequate vascularization to nutritionally support transplanted hepatocytes. We recently reported that matrix-immobilized angiogenic growth factors, e.g., endothelial cell growth factor (ECGF), can induce a high degree of neovascularization. Using this technique, we explored the possibility of transplanting isolated fetal porcine hepatocytes to create liver tissue organoids at a specific subcutaneous site. We evaluated chitosan as a scaffold biomaterial because of its structural similarity to glycosaminoglycans; glycosaminoglycans play a critical role in cell attachment, differentiation, and morphogenesis. Freshly isolated fetal porcine hepatocytes (FPH) (viability greater than 97%) were cultured on modified chitosan scaffolds and transplanted into rat groin fat pads with or without ECGF-induced neovascularization. Cell density and attachment kinetics on chitosan were examined by scanning electron microscopy (SEM) and quantified using a flavianic acid binding assay. Hepatocyte viability and liver organoid formation were examined immunohistochemically. FPH transplanted without prior neovascularization died within 1 day post-transplantation. When transplanted after ECGF-induced neovascularization, FPH thrived for at least 2 weeks and formed liver tissue like structures. Immunohistochemical analysis revealed the presence of hepatocyte-specific cytokeratin staining as well as the presence of alpha-fetoprotein. Light microscopy and SEM revealed that FPH did not change their morphology after attachment to the chitosan surfaces. Thus, chitosan-based biomaterial surfaces have good hepatocyte attachment properties. However, extensive neovascularization is essential for hepatocyte survival and organoid formation. In the future, chitosan-based biomaterials may be useful as scaffolds for creating liver tissue organoids.     

Development, characterization, and use of a fetal skin cell bank for tissue engineering in wound healing

Wound healing in fetal skin is characterized by the absence of scar tissue formation, which is not dependent on the intrauterine environment and amniotic fluid. Fetal cells have the capacity of extraordinary expansion and we describe herein the development of a fetal skin cell bank where from one organ donation (2-4 cm2) it is possible to produce several hundred million fetal skin constructs of 9 x 12 cm2. Fetal cells grow three to four times more rapidly than older skin cells cultured in the same manner and these banked fetal cells are very resistant against physical and oxidative stress when compared to adult skin cells under the same culture conditions. They are up to three times more resistant to UVA radiation and two times more resistant towards hydrogen peroxide treatment. This mechanism may be of major importance for fetal cells when they are delivered to hostile wound environments. For fetal cell delivery to patients, cells were associated with a collagen matrix to form a three-dimensional construct in order to analyze the capacity of these cells for treating various wounds. We have seen that fetal cells can modify the repair response of skin wounds by accelerating the repair process and reducing scarring in severe bums and wounds of various nature in children. Hundreds of thousands of patients could potentially be treated for acute and chronic wounds from one standardized and controlled cell bank.     

Human mesenchymal stem cell implantation and collagen modification as a tool for tissue engineering

Tissue engineering, in all its aspects, is a focus of increasing interest. Optimum vascularization has to be ensured ahead of transferring experimental designs to clinical applications. Our group searched for matrix modifications and cell sources liable to increase vessel formation in engineered tissue. Notwithstanding the ethical issues, adult human mesenchymal stem cells (hMSC) seemed to offer as unlimited a possibility of proliferation and differentiation as embryonic stem cells. These series of experiments focused on the differentiation capability of adult stem cells and organisation in modified collagen sponges. Adult mesenchymal stem cells were cultured to the second generation. Two different groups were formed: Group A cells remained in medium without additional growth factors. Group B cells were cultured in angiogenic differentiation medium. On day 21, the cells of both groups were detached and implanted into different collagen sponges: unmodified collagen sponges and EDC/NHS [1-ethyl-3(3-dimethyl-aminopropyl) carbodiimide (EDC) and N-hydroxysuccinmide (NHS)] cross-linked and heparinized collagen scaffolds. Twenty-one days later, the sponges were prepared for the histological evaluation. After 3 weeks in culture, the cells of group B showed phenotypic endothelial cell characteristics and expressed von-Willebrand factor. Enhanced proliferation and invasion on cross-linked and heparinised matrices were obtained with both cell types (A and B). In addition, the stem cell derived endothelial cells formed organised structures throughout the scaffolds. Obtained data revealed the differentiation capability of adult MSC into endothelial cells and organisation in modified collagen matrices. This approach might lead to optimised vascular incorporation in tissue replacement.M. Markowicz, A. Heitland have contributed equally in this work.     

Postnatal myocardial augmentation with skeletal myoblast-based fetal tissue engineering

BACKGROUND: Cardiac anomalies constitute the most common birth defects, many of which involve variable myocardial deficiencies. Therapeutic options for structural myocardial repair remain limited in the neonatal population. This study was aimed at determining whether engineered fetal muscle constructs undergo milieu-dependent transdifferentiation after cardiac implantation, thus becoming a potential means to increase/support myocardial mass after birth. METHODS: Myoblasts were isolated from skeletal muscle specimens harvested from fetal lambs, labeled by transduction with a retrovirus-expressing green fluorescent protein, expanded in vitro, and then seeded onto collagen hydrogels. After birth, animals underwent autologous implantation of the engineered constructs (n = 8) onto the myocardium as an onlay patch. Between 4 and 30 weeks postoperatively, implants were harvested for multiple analyses. RESULTS: Fetal and postnatal survival rates were 89% and 100%, respectively. Labeled cells were identified within the implants at all time points by immunohistochemical staining for green fluorescent protein. At 24 and 30 weeks postimplantation, donor cells double-stained for green fluorescent protein and Troponin I, while losing skeletal (type II) myosin expression. CONCLUSIONS: Fetal skeletal myoblasts engraft in native myocardium up to 30 weeks after postnatal, autologous implantation as components of engineered onlay patches. These cells also display evidence of time-dependent transdifferentiation toward a cardiomyocyte-like lineage. Further analysis of fetal skeletal myoblast-based constructs for the repair of congenital myocardial defects is warranted.     

Strategies for cell engineering in tissue repair

Cellular and tissue engineering are new areas of research, currently attracting considerable interest because of the remarkable potential they have for clinical application. Some claims have indeed been dramatic, including the possibility of growing complete, artificial organs, such as the liver. However, amid such long-term aspirations there is the very real possibility that small tissues (artificial grafts) may be fabricated in the near future for use in reconstructive surgery. Logically, we should focus on how it is possible to produce modest, engineered tissues for tissue repair. It is evident that strategies to date either depend on innate information within implanted cells, to reform the target tissue or aim to provide appropriate environmental cues or guidance to direct cell behavior. It is argued here that present knowledge of tissue repair biology points us toward the latter approach, providing external cues which will direct how cells should organize the new tissue. This will be particularly true where we need to reproduce microscopic and ultrastructural features of the original tissue architecture. A number of such cues have been identified, and methods are already available, including substrate chemistry, substrate contact guidance, mechanical loading, and biochemical mediators to provide these cues. Examples of these are already being used with some success to control the formation of tissue structures.     

Improving cell engraftment with tissue engineering

Cardiac cell therapy has not yet resulted in long-term clinical benefits or major recovery of myocardial function in humans. To date, most of the cardiac effects of cell-based therapy are believed to be mediated by a local angiogenic response rather than by the formation of neosyncytial contractile units such as had initially been hoped for. Therefore, repopulation of the ischemic or infarcted heart with progenitor cells that have vasculogenic potential may be an important mechanism to improve contractile function, both in the presence of viable and nonviable myocardium. This constitutes a focus within scientific reach; however, the low engraftment and viability of progenitor cells after transplantation necessitate the exploration of novel delivery techniques. Because biomaterials have the capacity to improve cell retention, survival, and differentiation, tissue engineering is now being explored as an approach to support cell-based therapies and enhance their efficacy. In this article, we address current progress made in tissue engineering to support cell therapy for the heart, and summarize our work in the development of biomaterials toward improving cell delivery and vascularization of ischemic tissue.     

组织工程软骨种子细胞研究进展

组织工程是应用生命科学和工程学原理,研究开发能够修复、维持或改善组织损伤能力的生物替代物的一门学科[1]。利用组织工程方法再造软骨,为临床上软骨缺损的修复带来了新的途径,种子细胞作为软骨组织工程学研究的首要方面,它的来源途径也就尤为重要。目前的组织工程软骨种子细胞的来源主要为自体软骨细胞、同种异体软骨细胞、成纤     

组织工程软骨种子细胞研究进展

组织工程是应用生命科学和工程学原理,研究开发能够修复、维持或改善组织损伤能力的生物替代物的一门学科[1]。利用组织工程方法再造软骨,为临床上软骨缺损的修复带来了新的途径,种子细胞作为软骨组织工程学研究的首要方面,它的来源途径也就尤为重要。目前的组织工程软骨种子细胞的来源主要为自体软骨细胞、同种异体软骨细胞、成纤维细胞、干细胞、转基因细胞等,成熟软骨细胞是最常被关注     

骨组织工程中种子细胞的选择

组织工程学是把材料学和生命学科有机结合的一门现代学科，材料学和细胞学的协同发展有效的促进了组织工程学的发展，材料载体学已取得了相当的发展，学者们面临着如何在骨组织工程中选取高效、便捷的种子，使工程化骨有效修复治疗骨缺损等骨骼疾病。细胞本文仅就组织工程学中种子细胞的发展一简要综述，以供读者参考。     

Decellularized vein as a potential scaffold for vascular tissue engineering

PURPOSE: Current strategies to create small-diameter vascular grafts involve seeding biocompatible, compliant scaffolds with autologous vascular cells. Our purpose was to study the composition and strength of decellularized vein to determine its potential as a vascular tissue-engineering scaffold. METHODS: Intact human greater saphenous vein specimens were decellularized by using sodium dodecyl sulfate (SDS). Residual cellular and extracellular matrix composition was studied with light and electron microscopy as well as immunohistochemistry. Burst and suture-holding strength was measured in vitro by insufflation and pull-through techniques. To assess initial handling and durability of decellularized vein in vivo, a canine model was developed wherein decellularized canine jugular veins were implanted as carotid interposition grafts in recipient animals. After two weeks of arterial perfusion, these grafts were studied with duplex imaging and histologic methods. RESULTS: Human saphenous vein decellularized by using SDS was devoid of endothelial cells and >94% of the cells resident within the vein wall. Collagen morphology appeared unchanged, and elastin staining decreased only slightly. Basement membrane collagen type IV remained intact. Compared with fresh vein, decellularized vein had similar in vitro burst (2480 +/- 460 mm Hg vs 2380 +/- 620 mm Hg; P >.05) and suture-holding (185 +/- 30 gm vs 178 +/- 66 gm; P >.05) strength. Decellularized canine vein functioned well in vivo without dilation, anastomotic complication, or rupture over 2 weeks of arterial perfusion. CONCLUSIONS: Vein rendered acellular with SDS has well-preserved extracellular matrix, basement membrane structure, and strength sufficient for vascular grafting. These properties suggest proof of concept for its use as a scaffold for further vascular tissue engineering. CLINICAL RELEVANCE: The following research examines the creation of a new small-diameter bypass graft. It is clinically relevant to patients who need distal arterial bypass, coronary artery bypass, or hemodialysis access, but who do not have adequate autologous vein for their surgeries. Future investigations will involve further tissue engineering of this vascular scaffold (eg, autologous endothelial seeding of its lumen) and testing the clinical usefulness of the completed graft.