Optimization of the culturing conditions of human umbilical cord blood-derived endothelial colony-forming cells under xeno-free conditions applying a transcriptomic approach.

Establishment of fetal bovine serum (FBS)-free cell culture conditions is essential for transplantation therapies. Blood-derived endothelial colony-forming cells (ECFCs) are potential candidates for regenerative medicine applications. ECFCs were isolated from term umbilical cord blood units and characterized by flow cytometry, capillary formation and responsiveness to cytokines. ECFCs were expanded under standard, FBS-containing endothelial medium, or transferred to chemically defined endothelial media without FBS. Microarray expression profiling was applied to compare the transcriptome profiles in FBS-containing versus FBS-free culture. ECFC outgrowth in standard medium was successful in 92% of cord blood units. The karyotype of expanded ECFCs remained normal. Without FBS, ECFC initiation and expansion failed. Modest proliferation, changes in cell morphology and organization and cell death have been observed after passaging. Gene ontology analysis revealed a broad down-regulation of genes involved in cell cycle progression and up-regulation of genes involved in stress response and apoptosis. Interestingly, genes participating in lipid biosynthesis were markedly up-regulated. Detection of several endothelial cell-specific marker genes showed the maintenance of the endothelial cell characteristics during serum-free culture. Although ECFCs maintain their endothelial characteristics during serum-free culturing, they could not be expanded. Additional supply of FBS-free media with lipid concentrates might increase the ECFC survival.

Multipotent mesenchymal stem cells from human placenta: critical parameters for isolation and maintenance of stemness after isolation.

OBJECTIVE: This study was undertaken to isolate and characterize multipotent mesenchymal stem cells from term human placenta (placenta-derived mesenchymal stem cells, PD-MSCs). STUDY DESIGN: Sequential enzymatic digestion was used to isolate PD-MSCs in which trypsin removes the trophoblast layer, followed by collagenase treatment of remaining placental tissue. Karyotype, phenotype, growth kinetics, and differentiability of PD-MSC isolates from collagenase digests were analyzed. RESULTS: PD-MSC isolation was successful in 14 of 17 cases. Karyotyping of PD-MSC isolates from deliveries with a male fetus revealed that these cells are of maternal origin. Flow cytometry and immunocytochemistry confirmed the mesenchymal stem cell phenotype. Proliferation rates of PD-MSCs remained constantly high up to passage 20. These cells could be differentiated toward mesodermal lineage in vitro up to passage 20. Nonconfluent culture was critical to maintain the MSC stemness during long-term culture. CONCLUSION: Term placenta constitutes a rich, very reliable source of maternal mesenchymal stem cells that remain differentiable, even at high passage numbers.

Injectable candidate sealants for fetal membrane repair: bonding and toxicity in vitro.

OBJECTIVE: This study was undertaken to test injectable surgical sealants that are biocompatible with fetal membranes and that are to be used eventually for the closure of iatrogenic membrane defects. STUDY DESIGN: Dermabond (Ethicon Inc, Norderstedt, Germany), Histoacryl (B. Braun GmbH, Tuttlingen, Germany), and Tissucol (Baxter AG, Volketwil, Switzerland) fibrin glue, and 3 types of in situ forming poly(ethylene glycol)-based polymer hydrogels were tested for acute toxicity on direct contact with fetal membranes for 24 hours. For the determination of elution toxicity, extracts of sealants were incubated on amnion cell cultures for 72 hours. Bonding and toxicity was assessed through morphologic and/or biochemical analysis. RESULTS: Extracts of all adhesives were nontoxic for cultured cells. However, only Tissucol and 1 type of poly(ethylene glycol)-based hydrogel, which is a mussel-mimetic tissue adhesive, showed efficient, nondisruptive, nontoxic bonding to fetal membranes. Mussel-mimetic tissue adhesive that was applied over membrane defects that were created with a 3.5-mm trocar accomplished leak-proof closure that withstood membrane stretch in an in vitro model. CONCLUSION: A synthetic hydrogel-type tissue adhesive that merits further evaluation in vivo emerged as a potential sealing modality for iatrogenic membrane defects.

Concise review: isolation and characterization of cells from human term placenta: outcome of the first international Workshop on Placenta Derived Stem Cells.

Placental tissue draws great interest as a source of cells for regenerative medicine because of the phenotypic plasticity of many of the cell types isolated from this tissue. Furthermore, placenta, which is involved in maintaining fetal tolerance, contains cells that display immunomodulatory properties. These two features could prove useful for future cell therapy-based clinical applications. Placental tissue is readily available and easily procured without invasive procedures, and its use does not elicit ethical debate. Numerous reports describing stem cells from different parts of the placenta, using nearly as numerous isolation and characterization procedures, have been published. Considering the complexity of the placenta, an urgent need exists to define, as clearly as possible, the region of origin and methods of isolation of cells derived from this tissue. On March 23-24, 2007, the first international Workshop on Placenta Derived Stem Cells was held in Brescia, Italy. Most of the research published in this area focuses on mesenchymal stromal cells isolated from various parts of the placenta or epithelial cells isolated from amniotic membrane. The aim of this review is to summarize and provide the state of the art of research in this field, addressing aspects such as cell isolation protocols and characteristics of these cells, as well as providing preliminary indications of the possibilities for use of these cells in future clinical applications.

Stem cell-like human endothelial progenitors show enhanced colony-forming capacity after brief sevoflurane exposure: preconditioning of angiogenic cells by volatile anesthetics.

BACKGROUND: Endothelial progenitor cells play a pivotal role in tissue repair, and thus are used for cell replacement therapies in "regenerative medicine." We tested whether the anesthetic sevoflurane would modulate growth or mobilization of these angiogenic cells. METHODS: In an in vitro model, mononuclear cells isolated from peripheral blood of healthy donors were preconditioned with sevoflurane (3 times 30 min at 2 vol% interspersed by 30 min of air). Colony-forming units were determined after 9 days in culture and compared with time-matched untreated control. Using magnetic cell sorting, CD133+/CD34+ endothelial progenitors were enriched from human umbilical cord blood, and vascular endothelial growth factor (VEGF), VEGFR2 (KDR), granulocyte colony-stimulating factor (G-CSF), STAT3, c-kit, and CXCR4 expressions were determined in sevoflurane-treated and untreated cells by real-time reverse transcriptase polymerase chain reaction. In a volunteer study with crossover design, we tested whether sevoflurane inhalation (<1 vol% end-tidal concentration) would mobilize endothelial progenitor cells from the bone marrow niche into the circulation using flow cytometry of peripheral blood samples. VEGF and G-CSF plasma levels were also measured. RESULTS: In vitro sevoflurane exposure of mononuclear cells enhanced colony-forming capacity and increased VEGF mRNA levels in CD133+/CD34+ cord blood cells (P = 0.017). Sevoflurane inhalation in healthy volunteers did not alter the number of CD133+/CD34+ or KDR+/CD34+ endothelial progenitors in the circulation, but increased the number of colony-forming units (P = 0.034), whereas VEGF and G-CSF plasma levels remained unchanged. CONCLUSIONS: Sevoflurane preconditioning promotes growth and proliferation of stem cell-like human endothelial progenitors. Hence, it may be used to promote perioperative vascular healing and to support cell replacement therapies.

EphA4 signaling regulates phospholipase Cgamma1 activation, cofilin membrane association, and dendritic spine morphology.

Specialized postsynaptic structures known as dendritic spines are the primary sites of glutamatergic innervation at synapses of the CNS. Previous studies have shown that spines rapidly remodel their actin cytoskeleton to modify their shape and this has been associated with changes in synaptic physiology. However, the receptors and signaling intermediates that restructure the actin network in spines are only beginning to be identified. We reported previously that the EphA4 receptor tyrosine kinase regulates spine morphology. However, the signaling pathways downstream of EphA4 that induce spine retraction on ephrin ligand binding remain poorly understood. Here, we demonstrate that ephrin stimulation of EphA4 leads to the recruitment and activation of phospholipase Cgamma1 (PLCgamma1) in heterologous cells and in hippocampal slices. This interaction occurs through an Src homology 2 domain of PLCgamma1 and requires the EphA4 juxtamembrane tyrosines. In the brain, PLCgamma1 is found in multiple compartments of synaptosomes and is readily found in postsynaptic density fractions. Consistent with this, PLC activity is required for the maintenance of spine morphology and ephrin-induced spine retraction. Remarkably, EphA4 and PLC activity modulate the association of the actin depolymerizing/severing factor cofilin with the plasma membrane. Because cofilin has been implicated previously in the structural plasticity of spines, this signaling may enable cofilin to depolymerize actin filaments and restructure spines at sites of ephrin-EphA4 contact.

The role of actively released fibrin-conjugated VEGF for VEGF receptor 2 gene activation and the enhancement of angiogenesis.

A major challenge for therapeutic delivery of angiogenic agents such as vascular endothelial growth factor (VEGF) is to achieve sustained, low dose signaling leading to durable neovessel formation. To this end, we recently created a variant of VEGF(121), TG-VEGF(121) that directly binds to fibrin and gets released locally in proteolysis-triggered manner. Here we combined noninvasive biophotonic monitoring of VEGF receptor 2 gene activation in transgenic VEGFR2-luc mice and histomorphometry to compare endothelial activation and long-term neovascularization by actively released TG-VEGF(121)versus passively released, diffusible wild-type VEGF(121) in subcutaneous fibrin implants. Monitoring in real-time over 3 weeks of luciferase signal driven by the VEGFR2 promoter revealed endothelial activation in skin exposed to wild-type VEGF(121), but no detectable elevation over fibrin alone by TG-VEGF(121). Histology at 3 weeks, however, demonstrated that TG-VEGF(121) promoted vessel growth significantly more effectively and reliably than wild-type VEGF(121). The majority of vessels surviving to 3 weeks contained stabilizing smooth muscle cells. Yet, by 6 weeks, no extra vessels induced by exogenous VEGF were left. In conclusion, release of fibrin-conjugated variant TG-VEGF(121) elicited lower VEGFR2-luc activation than wild-type VEGF(121) yet significantly more vascularization. In the absence of true physiological demand, even stabilized vessels are ultimately regressed.

The dosage dependence of VEGF stimulation on scaffold neovascularisation.

Growth factors are often used in tissue regeneration to stimulate vascularisation of polymeric scaffolds, with vascular endothelial growth factor (VEGF) having been extensively studied for short-term vessel ingrowth. We have therefore evaluated the effect of different concentrations of VEGF on the vascularisation of a porous scaffold in the short-, intermediate- and long-term, by delivering 15, 150 and 1500ng VEGF/day to polyurethane scaffolds by osmotic pumps for up to 6 weeks. An increased vascularisation months after termination of VEGF delivery was only achieved with 150ng/day (46%, p<0.05). This dosage consistently showed elevated levels of vascularisation (144, 125, 160 and 60% above PBS controls at 10, 20, 30 and 42 days, respectively, p<0.05), whilst the vessels induced by the highest dosage, though initially maximally elevated (265 and 270% at 10 and 20 days, p<0.05) tended to regress after 20 days of VEGF delivery. Pericyte coverage was decreased at 20 days for the highest dosage (30%, p<0.05). Lectin perfusion demonstrated that vessels within the scaffold were connected to the host vasculature at all time points and perfusion was substantially raised by VEGF delivery at day 20. These results suggest concentration of VEGF plays a critical role in the nature and persistence of vasculature formed in a tissue regenerative scaffold.

Comparative characterization of cultured human term amnion epithelial and mesenchymal stromal cells for application in cell therapy.

Emerging evidence suggests human amnion tissue as a valuable source of two distinct types of pluripotent cells, amnion epithelial cells (hAECs) and mesenchymal stromal cells (hAMSCs), for applications in cell replacement therapy. For some approaches, it may be necessary to culture and differentiate these cells before they can be transplanted. No systematic attempt has been yet made to determine the quantity and quality of amnion cells after isolation and culture. We looked at amnion cell isolates from 27 term placentas. Following our optimized protocol, primary yields were 6.3 x 10(6) hAECs and 1.7 x 10(6) hAMSCs per gram amnion. All 27 cases gave vital cultures of hAMSCs, while one third of cases (9 of 27) failed to give adherent cultures of hAECs. Primary cultures contained significantly more proliferating than apoptotic cells (hAECs: 16.4% vs. 4.0%; hAMSCs: 9.5% vs. 2.4%). Neither hAECs nor hAMSCs were clonogenic. They showed slow proliferation that almost stopped beyond passage 5. Microscopic follow-up revealed that hAEC morphology gradually changed towards mesenchymal phenotype over several passages. Flow cytometric characterization of primary cultures showed expression of mesenchymal progenitor markers CD73, CD90, CD105, and CD166, as well as the embryonic stem cell markers SSEA-3 and -4 on both amnion cell types. These profiles were grossly maintained in secondary cultures. Reverse transcriptase-PCR analysis exhibited transcripts of Oct-3/4 and stem cell factor in primary and secondary cultures of all cases, but no telomerase reverse transcriptase. Immunocytochemistry confirmed translation into Oct-3/4 protein in part of hAEC cultures, but not in hAMSCs. Further, both amnion cell types stained for CD90 and SSEA-4. Osteogenic induction studies with amnion cells from four cases showed significantly stronger differentiation of hAECs than hAMSCs; this capacity to differentiate greatly varied between cases. In conclusion, hAECs and hAMSCs in culture exhibit and maintain a similar marker profile of mesenchymal progenitors. hAECs were found as a less reliable source than hAMSCs and altered morphology during subculture.

Bioengineering of foetal membrane repair.

Preterm premature rupture of the foetal membranes (früher vorzeitiger Blasensprung) has remained a devastating complication of pregnancy with very high risk of pregnancy loss. Several methods of sealing spontaneously ruptured membranes to stop amniotic fluid leakage and prolong pregnancy have been tested, but no one of them has achieved a clinical breakthrough. Also, needle and foetoscopic puncture of membranes for diagnostic or surgical interventions in the amniotic cavity carry a significant risk of persistent membrane leakage and subsequent rupture - thus limiting the developing field of intrauterine foetal surgery. Efforts are concentrated on taking action before rupture rather than reacting after rupture: one avenue of research concerns prophylactic plugging of foetoscopic access sites in foetal membranes at the time of intervention, thus inhibiting leakage and rupture. Foetal membrane injuries, spontaneous or iatrogenic, constitute extreme challenges to repair: thinness of foetal membrane tissue, difficult localisation and accessibility of the rupture site, the need for injectable sealants, wet gluing conditions and poor wound healing in this tissue all complicate repair. The goal is to achieve immediate and at the same time long-lasting closure of the membrane leak. Here we review approaches to closure of foetal membrane defects with liquid sealants or solid biomaterial scaffolds, with the focus on prophylactic plugging of foetoscopic access sites.

Enzymatic formation of modular cell-instructive fibrin analogs for tissue engineering.

The molecular engineering of cell-instructive artificial extracellular matrices is a powerful means to control cell behavior and enable complex processes of tissue formation and regeneration. This work reports on a novel method to produce such smart biomaterials by recapitulating the crosslinking chemistry and the biomolecular characteristics of the biopolymer fibrin in a synthetic analog. We use activated coagulation transglutaminase factor XIIIa for site-specific coupling of cell adhesion ligands and engineered growth factor proteins to multiarm poly(ethylene glycol) macromers that simultaneously form proteolytically sensitive hydrogel networks in the same enzyme-catalyzed reaction. Growth factor proteins are quantitatively incorporated and released upon cell-derived proteolytic degradation of the gels. Primary stromal cells can invade and proteolytically remodel these networks both in an in vitro and in vivo setting. The synthetic ease and potential to engineer their physicochemical and bioactive characteristics makes these hybrid networks true alternatives for fibrin as provisional drug delivery platforms in tissue engineering.

Fetoscopic closure of punctured fetal membranes with acellular human amnion plugs in a rabbit model.

OBJECTIVE: To explore a surgical plug formed from decellularized term human amnion membrane for fetoscopic closure of iatrogenic defects in fetal membranes in a rabbit model. METHODS: The study was performed in eight rabbit does. Punctures were created at midgestational day 23 by 14-gauge needle fetoscopy on surgically exposed rabbit amniotic sacs. The entry sites were fetoscopically plugged either with decellularized term human amnion membrane (n=10) or previously successful commercial collagen matrix foil (n=10), followed by their primary fixation with fibrin glue and myometrial suturing. Seven punctured sacs without any plugging and 31 sacs without any manipulation served as two reference groups. Amniotic integrity and fetal parameters were assessed at gestational day 30. RESULTS: We established a facile method to prepare sheets of decellularized term human amnion membrane and verified its nontoxicity and cell compatibility in vitro. Decellularized term human amnion membrane sheets could be delivered precisely and controlled by fetoscopy as compact plugs into amniotic defects. The surgical handling characteristics of decellularized term human amnion membrane were better than the commercial collagen matrix foil. Treatment with human decellularized term human amnion membrane was comparable to treatment with the collagen matrix with regard to efficiency in restoring amniotic integrity. Seventy-five percent and 71.4% of amniotic sacs treated with decellularized term human amnion membrane or the commercial collagen matrix foil, respectively, showed amniotic integrity, compared with 25% in the left-open study group. Histology at the 1 week experimental endpoint showed no evidence for inflammation or beginning of anatomic healing of grafted, decellularized term human amnion membrane. CONCLUSION: Fetoscopic delivery of plugs made of decellularized term human amnion membrane presents a potentially practical surgical method to restore amniotic integrity of punctured fetal membranes. LEVEL OF EVIDENCE: III.

Cell-demanded liberation of VEGF121 from fibrin implants induces local and controlled blood vessel growth.

Although vascular endothelial growth factor (VEGF) has been described as a potent angiogenic stimulus, its application in therapy remains difficult: blood vessels formed by exposure to VEGF tend to be malformed and leaky. In nature, the principal form of VEGF possesses a binding site for ECM components that maintain it in the immobilized state until released by local cellular enzymatic activity. In this study, we present an engineered variant form of VEGF, alpha2PI1-8-VEGF121, that mimics this concept of matrix-binding and cell-mediated release by local cell-associated enzymatic activity, working in the surgically-relevant biological matrix fibrin. We show that matrix-conjugated alpha2PI1-8-VEGF121 is protected from clearance, contrary to native VEGF121 mixed into fibrin, which was completely released as a passive diffusive burst. Grafting studies on the embryonic chicken chorioallantoic membrane (CAM) and in adult mice were performed to assess and compare the quantity and quality of neovasculature induced in response to fibrin implants formulated with matrix-bound alpha2PI1-8-VEGF121 or native diffusible VEGF121. Our CAM measurements demonstrated that cell-demanded release of alpha2PI1-8-VEGF121 increases the formation of new arterial and venous branches, whereas exposure to passively released wild-type VEGF121 primarily induced chaotic changes within the capillary plexus. Specifically, our analyses at several levels, from endothelial cell morphology and endothelial interactions with periendothelial cells, to vessel branching and network organization, revealed that alpha2PI1-8-VEGF121 induces vessel formation more potently than native VEGF121 and that those vessels possess more normal morphologies at the light microscopic and ultrastructural level. Permeability studies in mice validated that vessels induced by alpha2PI1-8-VEGF121 do not leak. In conclusion, cell-demanded release of engineered VEGF121 from fibrin implants may present a therapeutically safe and practical modality to induce local angiogenesis.

Conditional human VEGF-mediated vascularization in chicken embryos using a novel temperature-inducible gene regulation (TIGR) system.

Advanced heterologous transcription control systems for adjusting desired transgene expression are essential for gene function assignments, drug discovery, manufacturing of difficult to produce protein pharmaceuticals and precise dosing of gene-based therapeutic interventions. Conversion of the Streptomyces albus heat shock response regulator (RheA) into an artificial eukaryotic transcription factor resulted in a vertebrate thermosensor (CTA; cold-inducible transactivator), which is able to adjust transcription initiation from chimeric target promoters (P(CTA)) in a low-temperature- inducible manner. Evaluation of the temperature-dependent CTA-P(CTA) interaction using a tailored ELISA-like cell-free assay correlated increased affinity of CTA for P(CTA) with temperature downshift. The temperature-inducible gene regulation (TIGR) system enabled tight repression in the chicken bursal B-cell line DT40 at 41 degrees C as well as precise titration of model product proteins up to maximum expression at or below 37 degrees C. Implantation of microencapsulated DT40 cells engineered for TIGR-controlled expression of the human vascular endothelial growth factor A (hVEGF121) provided low-temperature-induced VEGF-mediated vascularization in chicken embryos.

Adult 'endothelial progenitor cells'. Renewing vasculature.

During embryogenesis, endothelial progenitor cells participate in the initial processes of primitive blood vessel formation (vasculogenesis). It has become evident that progenitors to vascular endothelial cells also exist in the adult. Endothelial progenitors normally reside in the adult bone marrow but may become mobilized into circulation by cytokine or angiogenic growth factor signals from the periphery, enter extravascular tissue, and promote de novo vessel formation by virtue of physically integrating into vessels and/or supplying growth factors (adult vasculogenesis). For that reason, autologous endothelial progenitors, mobilized in situ or transplanted, has become a major target of therapeutic revascularization approaches to ischemic disease and endothelial injury. Moreover, endothelial progenitors represent a potential target of strategies to block tumor growth.

Cell-demanded release of VEGF from synthetic, biointeractive cell ingrowth matrices for vascularized tissue growth.

Local, controlled induction of angiogenesis remains a challenge that limits tissue engineering approaches to replace or restore diseased tissues. We present a new class of bioactive synthetic hydrogel matrices based on poly(ethylene glycol) (PEG) and synthetic peptides that exploits the activity of vascular endothelial growth factor (VEGF) alongside the base matrix functionality for cellular ingrowth, that is, induction of cell adhesion by pendant RGD-containing peptides and provision of cell-mediated remodeling by cross-linking matrix metalloproteinase substrate peptides. By using a Michael-type addition reaction, we incorporated variants of VEGF121 and VEGF165 covalently within the matrix, available for cells as they invade and locally remodel the material. The functionality of the matrix-conjugated VEGF was preserved and was critical for in vitro endothelial cell survival and migration within the matrix environment. Consistent with a scheme of locally restricted availability of VEGF, grafting of these VEGF-modified hydrogel matrices atop the chick chorioallontoic membrane evoked strong new blood vessel formation precisely at the area of graft-membrane contact. When implanted subcutaneously in rats, these VEGF-containing matrices were completely remodeled into native, vascularized tissue. This type of synthetic, biointeractive matrix with integrated angiogenic growth factor activity, presented and released only upon local cellular demand, could become highly useful in a number of clinical healing applications of local therapeutic angiogenesis.

Endothelial cell proliferation and progenitor maturation by fibrin-bound VEGF variants with differential susceptibilities to local cellular activity.

A number of vascular therapies could benefit from advanced methods for presentation of angiogenic growth factors, including growth of endothelium on small caliber vascular grafts and revascularization of ischemic tissue through induction of collateral vessels and microvessels. To explore methods to optimize the presentation and release of angiogenic factors in such applications in device integration and tissue repair, we studied three variant forms of vascular endothelial growth factor 121 (VEGF121), each with differential susceptibility to local cellular proteolytic activity, formulated within fibrin matrices. (1) The prototypic variant alpha2PI(1-8)-VEGF121 remains immobilized in fibrin matrices until its liberation by cell-associated enzymes, such as plasmin, that degrade the fibrin network [slow, cell-demanded release; J. Control. Release 72 (2001) 101-113]; the alpha2PI(1-8) domain serves as a site for covalent attachment to fibrin during coagulation. (2) We created a new VEGF variant, alpha2PI(1-8)-Pla-VEGF121 that couples to fibrin via a plasmin-sensitive sequence (Pla). Cleavage of this target site by plasmin enables direct release of alpha2PI(1-8)-Pla-VEGF121 from bulk matrix degradation (accelerated, cell-demanded release). (3) Native VEGF121 (burst, passive release) was considered as a reference. VEGF release profiles were determined experimentally as well as mathematically, alpha2PI(1-8)-Pla-VEGF121 being released ca. fourfold more quickly than alpha2PI(1-8)-VEGF121, both being retained compared to native VEGF121; the differences in release could be accounted for based on knowledge of the plasmin sensitivity of the bound growth factor and the structure of the fibrin network. The bound factors were competent in inducing endothelial cell proliferation, the matrix-bound forms being more effective than native VEGF121; as well as competent in inducing endothelial progenitor cell maturation into endothelial cells. These matrix-bound variants of VEGF121 may be particularly useful where retention in locally applied surgical sites is desired, such as prevention of washout from vascular graft coatings and slowing loss from tissue ingrowth matrices used in local tissue revascularization and repair.

Tissue engineering of angiogenesis with autologous endothelial progenitor cells.

Adult bone marrow and peripheral blood contain small subsets of mononuclear cells that can be differentiated into endothelial-like cells in vitro. Experimental and clinical transplantation of such cell isolates--often referred to as endothelial stem/progenitor cells--into ischaemic or infarcted areas shows their incorporation into sites of new vessel growth along with improvement of regional blood flow. Emerging evidence suggests that these beneficial effects on vascular growth can be attributed to the paracrine activation of resident endothelial cells, rather than their integration into new endothelium. Autologous endothelial progenitor cells can also substitute for native vessel-derived endothelial cells in tissue-engineered vascular autografts.

Engineered fibrin matrices for functional display of cell membrane-bound growth factor-like activities: study of angiogenic signaling by ephrin-B2.

With the rapid increase in approaches to pro- or anti-angiogenic therapy, new and effective methodologies for administration of cell-bound growth factors will be required. We sought to develop the natural hydrogel matrix fibrin as platform for extensive interactions and continuous signaling by the vascular morphogen ephrin-B2 that normally resides in the plasma membrane and requires multivalent presentation for ligation and activation of Eph receptors on apposing endothelial cell surfaces. Using fibrin and protein engineering technology to induce multivalent ligand presentation, a recombinant mutant ephrin-B2 receptor binding domain was covalently coupled to fibrin networks at variably high densities. The ability of fibrin-bound ephrin-B2 to act as ligand for endothelial cells was preserved, as demonstrated by a concomitant, dose-dependent increase of endothelial cell binding to engineered ephrin-B2-fibrin substrates in vitro. The therapeutic relevance of ephrin-B2-fibrin implant matrices was demonstrated by a local angiogenic response in the chick embryo chorioallontoic membrane evoked by the local and prolonged presentation of matrix-bound ephrin-B2 to tissue adjacing the implant. This new knowledge on biomimetic fibrin vehicles for precise local delivery of membrane-bound growth factor signals may help to elucidate specific biological growth factor function, and serve as starting point for development of new treatment strategies.

Biopolymeric delivery matrices for angiogenic growth factors.

The development of new therapeutic approaches that aim to help the body exert its natural mechanisms for vascularized tissue growth (therapeutic angiogenesis) has become one of the most active areas of tissue engineering. Through basic research, several growth factor families and cytokines that are capable to induce physiological blood vessel formation have been identified. Indeed, preclinical and clinical investigations have indicated that therapeutic administration of angiogenic factors, such as the prototypic vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF), to sites of ischemia in the heart or the limb can improve regional blood flow. For new and lasting tissue vascularization, prolonged tissue exposure to these factors could be critical. Furthermore, as shown for VEGF, dosage must be tightly controlled, as excess amounts of VEGF can cause severe vascular leakage and hypotension. This review emphasizes natural and synthetic polymer matrices with respect to their development as vehicles for local and controlled delivery of angiogenic proteins, such as VEGF and bFGF, and their clinical applicability. In the dawn of experimental vascular engineering, new biomaterial schemes for clinical growth factor administration that take better account of biological principles of angiogenic growth factor function and the cell biological basis necessary to produce functional vasculature are evolving. Alongside their base function as protective embedment for angiogenic growth factors, these new classes of bioactive polymers are engineered with additional functionalities that better preserve growth factor activity and more closely mimic the in vivo release mechanisms and profiles of angiogenic growth factors from the extracellular matrix (ECM). Consequently, the preparation of both natural or completely synthetic materials with biological characteristics of the ECM has become central to many tissue engineering approaches that aim to deliver growth factors in a therapeutically efficient mode. Another promising venue to improve angiogenic performance is presented by biomaterials that allow sequential delivery of growth factors with complementary roles in blood vessel initiation and stabilization.