Chorioallantoic membrane capillary bed: a useful target for studying angiogenesis and anti-angiogenesis in vivo.

The chick embryo chorioallantoic membrane (CAM) is an extraembryonic membrane that is commonly used in vivo to study both angiogenesis and anti-angiogenesis. This review 1) summarizes the current knowledge about the structure of the CAM's capillary bed; 2) discusses the controversy about the existence of a single blood sinus or a capillary plexus underlying the chorionic epithelium; 3) describes a new model of the CAM vascular growth, namely the intussusceptive mode; 4) reports findings regarding the role played by endogenous fibroblast growth factor-2 in CAM vascularization; and 5) addresses the use and limitations of the CAM as a model for studying angiogenesis and anti-angiogenesis.     

Chorioallantoic membrane capillary bed: A useful target for studying angiogenesis and anti-angiogenesis in vivo

The chick embryo chorioallantoic membrane (CAM) is an extraembryonic membrane that is commonly used in vivo to study both angiogenesis and anti-angiogenesis. This review 1) summarizes the current knowledge about the structure of the CAM's capillary bed; 2) discusses the controversy about the existence of a single blood sinus or a capillary plexus underlying the chorionic epithelium; 3) describes a new model of the CAM vascular growth, namely the intussusceptive mode; 4) reports findings regarding the role played by endogenous fibroblast growth factor-2 in CAM vascularization; and 5) addresses the use and limitations of the CAM as a model for studying angiogenesis and anti-angiogenesis. Anat Rec 264:317–324, 2001. © 2001 Wiley-Liss, Inc.     

Modulation of in vitro angiogenesis in a three-dimensional spheroidal coculture model for bone tissue engineering

One of the major challenges in tissue engineering of bone substitutes remains vascularization of the transplant. We have developed a three-dimensional collagen-based coculture system to assess interactions between human endothelial cells (hECs) and human osteoblasts (hOBs) in vitro. Human umbilical vein endothelial cells (HUVECs) were grown as three-dimensional multicellular spheroids and seeded in a collagen matrix to assess sprouting of the spheroids, that is, formation of tubelike structures resembling early capillaries. Direct cell contact between hOBs and HUVECs was established by incorporating hOBs into the EC spheroids, thus forming heterogeneous cospheroids. Spatial organization of cospheroids and sprout configuration were assessed by immunohistochemical wholemount staining techniques and confocal laser microscopy. Cumulative sprout length of spheroids was quantitatively analyzed by digital imaging planimetry. In this model HUVECs and hOBs formed heterogeneous cospheroids with distinct spatial organization. The ability of HUVEC spheroids to form tubelike structures on angiogenic stimulation with vascular endothelial growth factor and basic fibroblast growth factor was suppressed in heterogeneous HUVEC/hOB cospheroids. The model system introduced in this study may be useful to assess the mechanisms involved in regulating angiogenesis during bone formation and to further investigate the mechanisms by which heterotypic cell-cell interactions inhibit endothelial tube formation for applications in bone tissue engineering.     

Small molecule inducers of angiogenesis for tissue engineering

Engineering of implantable tissues requires rapid induction of angiogenesis to meet the significant oxygen and nutrient demands of cells during tissue repair. To this end, our laboratories have utilized medicinal chemistry to synthesize non-peptide-based inducers of angiogenesis to aid tissue engineering. In this study, we describe the evaluation of SC-3-149, a small molecule compound with proliferative effects on vascular endothelial cells. Specifically, exogenous exposure of SC-3-149 induced an 18-fold increase in proliferation of human microvascular endothelial cells in vitro at low micromolar potency by day 14 in culture. Moreover, SC-3-149 significantly increased the formation of endothelial cord and tubelike structures in vitro, and improved endothelial scratch wound healing within 24 h. SC-3-149 also significantly inhibited vascular endothelial cell death owing to serum deprivation and high acidity (pH 6). Concurrent incubation of SC-3-149 with vascular endothelial growth factor increased cell survivability under serum-deprived conditions by an additional 7%. In addition, in vivo injection of SC-3-149 into the rat mesentery produced qualitative increases in microvessel length density. Taken together, our studies suggest that SC-3-149 and its analogs may serve as promising new angiogenic agents for targeted drug delivery and therapeutic angiogenesis in tissue engineering.     

Angiogenic growth factor synergism in a murine tissue engineering model of angiogenesis and adipogenesis

De novo tissue generation stimulated by three angiogenic growth factors administered in a factorial design was studied in an in vivo murine tissue engineering chamber. A silicone chamber was implanted around the epigastric pedicle and filled with Matrigel with 100 ng/ml of recombinant mouse vascular endothelial growth factor-120 (VEGF120), recombinant human basic fibroblastic growth factor (FGF-2), or recombinant rat platelet-derived growth factor-BB (PDGF-BB) added as single, double, or triple combinations. Angiogenesis, supporting tissue ingrowth, and adipogenesis were assessed at 2 and 6 weeks by immunohistochemistry and morphometry. At 2 weeks angiogenesis was synergistically enhanced by VEGF120 + FGF-2 (P = 0.019). FGF-2 (P = 0.008) and PDGF-BB (P = 0.01) significantly increased connective tissue/inflammatory cell infiltrate (macrophages, pericytes, and preadipocytes) in double and triple combinations compared with control. At 6 weeks sequential addition of growth factors increased the percent volume of adipose tissue (P < 0.0005, each main effect), with a synergistic increase in adipose tissue in combination treatments (P < 0.0005). Groups containing 300 ng/ml of single growth factors produced significantly less adipose tissue than the triple growth factor combination (P < 0.0005, VEGF120 and PDGF-BB; P < 0.001, FGF-2). In conclusion, angiogenic growth factor combinations increased early angiogenesis and cell infiltration resulting in synergistically increased adipose tissue growth at 6 weeks. Two way and higher level synergies are likely to be important in therapeutic applications of angiogenic growth factors.     

Novel biodegradable electrospun membrane: scaffold for tissue engineering

Nonwoven fibrous matrixes have been widely used as scaffolds in tissue engineering, and modification of microstructure of these matrices is needed to organize cells in three-dimensional space with spatially balanced proliferation and differentiation required for functional tissue development. The objective of this study was fabrication of nanofibrous matrix from novel biodegradable poly(p-dioxanone-co-L-lactide)-block-poly(ethylene glycol) (PPDO/PLLA-b-PEG) copolymer, and to examine cell proliferation, morphology of cell-matrix interaction with the electrospun nanofibrous matrix. The electrospun structure composed of PPDO/PLLA-b-PEG fibers with an average diameters of 380 nm, median pore size 8 microm, porosity more than 80% and mechanical strength 1.4 MPa, is favorable for cell-matrix interaction and supports the active biocompatibility of the structure. NIH 3T3 fibroblast cell seeded on the structure tend to maintain phenotypic shape and guided growth according to nanofiber orientation. Good capability of the nanofibrous structure for supporting the cell attachment and proliferation are observed. This novel biodegradable scaffold will be applicable for tissue engineering based upon its unique architecture, which acts to support and guide cell growth.     

Heterochromatin assembly: A new twist on an old model

The organization of eukaryotic genomes requires a harmony between efficient compaction and accessibility. This is achieved through its packaging into chromatin. Chromatin can be subdivided into two general structural and functional compartments: euchromatin and heterochromatin. Euchromatin comprises most of the expressed genome, while heterochromatin participates intimately in the production of structures such as centromeres and telomeres essential for chromosome function. Studies in the fission yeast Schizosaccharomyces pombe have begun to highlight the genetic pathways critical for the assembly and epigenetic maintenance of heterochromatin, including key roles played by the RNAi machinery, H3 lysine 9 methylation and heterochromatin protein 1 (HP1). Recent studies have also identified a novel E3 ubiquitin ligase universally required for H3 K9 methylation. Here we outline these studies and propose several models for the role of this E3 ligase in heterochromatin assembly.     

Fast and efficient screening system for new biomaterials in tissue engineering: a model for peripheral nerve regeneration

The use of three-dimensional biodegradable matrices is one major issue in tissue engineering. Numerous materials, fabrication techniques, and modifications have been used and tested in different areas of tissue engineering recently. But nevertheless, technology is far from being optimized and optimal constructs with bioidentical and mechanical properties have not been described in the literature so far. Hence, there is great demand of new suitable biomaterials for tissue engineering applications. In this study, a fast and efficient screening system for initial testing of biomaterials for cell culture application was developed. The set up for the screening system and the decision criteria applied for the determination of suitability of new materials are presented. Hep-G2 and PC-12 cells were seeded onto different matrices and cultured over a period of 2 weeks. The viability of the cells was monitored via the MTT assay. Cell spreading was investigated by DAPI-staining of cell nuclei. Furthermore, the adhesion of the cells on the different matrices was examined by counting the number of attached cells. With these general assays a classification of materials is possible with regard to their suitability. Optimal cell models must be chosen for the defined applications and at least two cell lines are necessary for a differentiating interpretation.     

Development and characterization of a spheroidal coculture model of endothelial cells and fibroblasts for improving angiogenesis in tissue engineering

Neovascularization is a critical step in tissue engineering applications since implantation of voluminous grafts without sufficient vascularity results in hypoxic cell death of central tissues. We have developed a three-dimensional spheroidal coculture system consisting of human umbilical vein endothelial cells (HUVECs) and human primary fibroblasts (hFBs) to improve angiogenesis in tissue engineering applications. Morphological analysis of cryosections from HUVEC/hFB cospheroids revealed a characteristic temporal and spatial organization with HUVECs located in the center of the cospheroid and a peripheral localization of fibroblasts. In coculture spheroids, the level of apoptosis of endothelial cells was strongly decreased upon cocultivation with fibroblasts. Collagen-embedded HUVEC spheroids develop numerous lumenized capillary-like sprouts. This was also apparent for HUVEC/hFB cospheroids, albeit to a lesser extent. Quantification of cumulative sprout length revealed an approximately 35% reduction in endothelial cell sprouting upon cocultivation with fibroblasts in cospheroids. The slight reduction in endothelial cell sprouting was not mediated by a paracrine mechanism but is most likely due to the formation of heterogenic cell contacts between HUVECs and hFBs within the cospheroid. The model system introduced in this study is suitable for the development of a preformed lumenized capillary-like network ex vivo and may therefore be useful for improving angiogenesis in in vivo tissue engineering applications.     

Cardiovascular tissue engineering: a new laminar flow chamber for in vitro improvement of mechanical tissue properties

A new in vitro flow system was developed to investigate the impact of laminar flow on extracellular matrix formation and tissue development. The dynamic in vitro system was designed to provide a cross flow arrangement of main flow induced by a dialysis roller pump (500 ml/min), and nutrition flow by a perfusion pump (3 ml/hr). Poly-L-lysine precoated polyglycolic acid (PGA) scaffolds (3.14 cm2) were seeded with myofibroblasts of human aortic origin (3.0 x 10(6) cells/ mesh) and incubated for 14 days under static conditions. The tissue was exposed to shear stress over a time period of 14 days (n = 4). The control group was seeded under static conditions (n = 4). To obtain a CO2 independent medium, 25 mM HEPES and 1 mM bicarbonate buffer was supplemented to modified MEM without bicarbonate. Gas samples were collected from the medium, and hydroxyproline assay was performed as a marker of collagen production. The newly developed flow system maintained stable cell culture conditions, with the hydroxyproline concentration significantly higher in group F (p < 0.05). These preliminary experiences with a new in vitro tissue culture system demonstrate the feasibility of using flow induced mechanical stress to enhance extracellular matrix formation.     

Chorioallantoic membrane for in vivo investigation of tissue-engineered construct biocompatibility

In tissue engineering approach, the scaffold plays a key role for a suitable outcome of cell-scaffold interactions and for the success of tissue healing and regeneration. As a consequence, the characterization of scaffold properties and the in vivo evaluation of tissue responses and effects result to be essential in the development of suitable implantable device. Among the in vivo methods, the chick embryo chorioallantoic membrane (CAM) assay represents a rather simple and cost-effective procedure to study the biocompatibility responses of graft materials. CAM is indeed characterized by low experiment costs, simplicity, relative speed in obtaining the expected results, limited ethical concern, no need of high-level technical skill, and the absence of a mature immune system, resulting in an inexpensive, simple, and practical method to evaluate and characterize tissue-engineered constructs. The results till now obtained suggest that CAM assay can be used as a pre-screening assay, before in vivo animal studies, to determine whether the scaffold is liable to cause an adverse reaction and to evaluate its future enhancement of existing materials for tissue engineering. A review of the more recent results related to the use of CAM for in vivo biomaterial property evaluation is herein reported.     

Tissue-engineering bioreactors: a new combined cell-seeding and perfusion system for vascular tissue engineering

One approach to the tissue engineering of vascular structures is to develop in vitro conditions in order ultimately to fabricate functional vascular tissues before final implantation. In our experiment, we aimed to develop a new combined cell seeding and perfusion system that provides sterile conditions during cell seeding and biomechanical stimuli in order to fabricate autologous human vascular tissue in vitro. The cell seeding and perfusion system is made of Plexiglas and is completely transparent (Berlin Heart, Berlin, Germany; University Hospital Benjamin Franklin, Berlin, Germany). The whole system consists of a cell seeding chamber that can be incorporated into the perfusion system and an air-driven respirator pump connected to the bioreactor. The cell culture medium continuously circulates through a closed-loop system. We thus developed a cell seeding device for static and dynamic seeding of vascular cells onto a polymeric vascular scaffold and a closed-loop perfused bioreactor for long-term vascular conditioning. The cell seeding chamber can be easily connected to the bioreactor, which combines continuous, pulsatile perfusion and mechanical stimulation to the tissue-engineered conduit. Adjusting the stroke volume, the stroke rate, and the inspiration/expiration time of the ventilator allows various pulsatile flows and different levels of pressure. The whole system is a highly isolated cell culture setting, which provides a high level of sterility and a gas supply and fits into a standard humidified incubator. The device can be sterilized by ethylene oxide and assembled with a standard screwdriver. Our newly developed combination of a cell seeding and conditioning device provides sterile conditions and biodynamic stimuli for controlled tissue development and in vitro conditioning of an autologous tissue-engineered vessel.     

新型脊髓纳米组织工程支架的组织相容性

背景：纳米技术可改善脊髓组织工程生物材料的性能。 目的：分析新型脊髓纳米组织工程支架的组织相容性。 方法：以胶原为原料制备纤维定向排列及非定向排列的纳米纤维膜,培养及鉴定SD大鼠脊髓源性神经干细胞。将两种纳米纤维膜与SD乳鼠脊髓源性神经干细胞共培养,以正常培养的神经干细胞为对照,通过MTT实验检测纳米纤维膜的细胞相容性;以扫描电镜检测细胞在纳米纤维膜表面的黏附及增殖情况;将纳米纤维膜植入SD大鼠体内,通过组织学检查确定其降解情况及组织相容性;通过免疫组织化学实验确定神经干细胞在体内的存活及移动情况。 结果与结论：两种纳米纤维膜表面的神经干细胞黏附及增殖情况良好,MTT实验结果表明纳米纤维膜的细胞相容性佳,电镜结果表明细胞在纳米纤维膜表面黏附良好,增殖情况佳;在体内纳米纤维膜降解情况良好,组织相容性佳;BrdU标定的神经干细胞在SD大鼠体内存活并移动情况良好。结果表明新型纳米组织工程支架具有良好的细胞及组织相容性。     

Stem cells: new cell source for myocardial constructs tissue engineering

Cardiovascular diseases like myocardial infarction, complex congenital heart disease, and subsequent heart failure are a leading cause of morbidity and mortality. Recent advances in tissue engineering arise to address the lack of available tissues and organs for transplantation because cells alone are not capable of recreating complex tissues upon transplantation. Consequently, a very promising approach to repair large scar areas and congenital heart defects may be the use of tissue engineering, in which cells are seeded in three-dimensional matrices of biodegradable polymers to form myocardial constructs. In recent years, there has been a tremendous increase in the understanding of stem cell biology. Stem cells have clonogenic and self-renewing capabilities, and under certain conditions, can differentiate into multiple cell lineages. Recent studies have shown that stem cells can be isolated from a wide variety of tissues, including bone marrow, peripheral blood, muscle, and adipose tissue. We hypothesize that tissue-engineered myocardial constructs with stem cells may fulfill the requirements of native heart muscle and, in the long run, may allow replacement of the injured heart and repair of congenital cardiac defects possible.     

Promotion of angiogenesis in tissue engineering: developing multicellular matrices with multiple capacities

One of the aims of tissue engineering is to be able to develop multi-tissue organs in the future. This requires the optimization of conditions for the differentiation of multiple cell types and maintenance of the differentiated phenotype within complex engineered tissues. The goal of this study was to develop prototype tissue engineered matrices to support the simultaneous growth of different cell types with a particular focus on the angiogenic process. We examined two different matrix compositions for the promotion of blood vessel and tube formation. A fibrin-based matrix with the addition of a combination of growth factors supported vascular growth and the invasion of inflammatory cells. Using this fibrin matrix, in combination with a collagen-based hydrogel, a simple in vitro model of the cornea with adjacent sclera was developed that was complete with innervation and vascular structures. In addition, we showed that collagen-based matrices were effective in delivering mononuclear endothelial progenitor cells to ischemic tissue in vivo, and allowing these cells to incorporate into vascular structures. It is anticipated that with further development, these matrices have potential for use as delivery matrices for cell transplantation and for in vitro study purposes of multiple cell types.     

Fabrication of a new tubular fibrous PLCL scaffold for vascular tissue engineering

Biodegradable macroporous scaffolds have been developed for tissue-engineering applications. We fabricated and characterized a new tubular, macroporous, fibrous scaffold using a very elastic biodegradable co-polymer, poly(L-lactide-co-caprolactone) (PLCL, 5:5) in a gel-spinning process. A viscous PLCL solution was spun as a gel-phase under swirl-flow conditions and was subsequently fabricated to produce a tubular fibrous scaffold on a rotating cylindrical shaft in a methanol solution. The porosity and median pore size of the fibrous PLCL scaffolds were 55-75% and 120-150 microm, respectively, using a 5-10% PLCL solution. The use of a 7.5% (w/v) solution resulted in scaffolds with tensile strength and elastic modulus of 3.39 MPa and 1.22 MPa, respectively. The scaffolds exhibited 500-600% elongation-at-break. The tensile strength and modulus of fibrous PLCL scaffolds were proven to decrease on lowering the concentration of the PLCL spinning solution; however, the tensile strength and modulus of fibrous PLCL scaffolds, produced from 5% solutions, are approximately 4- and 5-times higher than those of extruded PLCL scaffolds. These properties indicated that the fibrous PLCL scaffolds were very elastic and mechanically strong. The scaffolds appeared to be well inter-connected between the pores as determined by SEM imaging analysis. In addition, the cell-seeding efficiency was 2-fold higher using gel-spun scaffolds than using extruded scaffolds. These results suggest that the gel-spun fibrous PLCL scaffold is an excellent matrix for vascular tissue-engineering applications.     

Application of magnetic resonance microscopy to tissue engineering: a polylactide model

Absorbable polymers are unique materials that find application as temporary scaffolds in tissue engineering. They are often extremely sensitive to histological processing and, for this reason, studying fragile, tissue-engineered constructs before implantation can be quite difficult. This research investigates the use of noninvasive imaging using magnetic resonance microscopy (MRM) as a tool to enhance the assessment of these cellular constructs. A series of cellular, polylactide constructs was developed and analyzed using a battery of tests, including MRM. Distribution of rat aortic smooth muscle cells within the scaffolds was compared as one example of a tissue engineering MRM application. Cells were loaded in varying amounts using static and dynamic methods. It was found that the cellular component was readily identified and the polymer microstructure readily assessed. Specifically, the MRM results showed a heterogeneous distribution of cells due to static loading and a homogenous distribution associated with dynamic loading, results that were not visible through biochemical tests, scanning electron microscopy, or histological evaluation independently. MRM also allowed differentiation between different levels of cellular loading. The current state of MRM is such that it is extremely useful in the refinement of polymer processing and cell seeding methods. This method has the potential, with technological advances, to be of future use in the characterization of cell-polymer interactions.