Three-dimensional Fibrin Matrix Research Articles

Platelet-rich preparations as biocarriers for local drugs for periodontal disease: A narrative review

Abstract Introduction: Platelets are nucleated cells that form blood clots as their primary function, but their popularity comes with secondary functions such as angiogenesis and innate immunity. Aims: This review focuses on different methods of platelet-rich plasma production and its use as a drug carrier, particularly in dental medicine. Materials and Methods: Scientific databases were searched from 2006 to 2013. Results and Discussion: Platelet-derived growth factors are released during the modulation of the wound-healing process and regulate many biological activities. High regeneration capacity in the wound healing process combines its use with traditional methods in medical applications and sometimes in alternative treatment methods. Platelet-rich biological structures have advantages such as rapid and easy production, antimicrobial properties, high regenerative capacity, and inhibition of postoperative bleeding. Platelet-rich autogenous blood concentrates can administer antibiotics and anti-inflammatory drugs locally. Platelet-rich biological structures can also be used as high-quality carriers for the targeted delivery of drugs. Conclusion: Apart from antibiotics, orally administered analgesics, anticancer drugs, etc., can also be added to platelet-rich fibrin because the three-dimensional fibrin matrix structure undergoes fibrinolysis completely autogenously within the body. Still, biocompatibility, biodegradability, mechanical strength, and shape retention must be investigated for optimal drug utilization.

P446 Plasma rich in growing factors as treatment for perianal fistulas in Crohn’s disease: unicentre experience

Abstract Background Perianal Crohn’s disease (PCD) adds a significant morbidity and reduced quality of life. Complex fistulas occur in up to 20% of patients. The available treatment of PCD includes immunosuppressive drugs, antibiotics, different surgeries but are associated with high recurrence rates. A new alternative using plasma rich in growth factors (PRGF) for the treatment of complex fistulas has emerged from to achieve better healing with the minimum possible complications without affecting anal continence. This study aims to assess the feasibility, safety and efficacy of local injection of PRGF in patients with PCD. Methods Retrospective observational and descriptive study analyzing 6 Crohn′s Disease (CD) patients undergoing complex anal fistula sealing with PRGF from March 2019 to December 2020. All patients underwent endoanal ultrasound and 60% a pelvic magnetic resonance also. The technique consisted of locating the tract without the use of hydrogen peroxide, active curettage of the tract and closure the internal fistulous orifice (IFO) with single stitches Vicryl® 3/0. Subsequently, 50% of the PRGF-rich fraction was injected in the submucosa of the closed internal fistula orifice. The other 50% is injected in the fistulous tract. With PRGF-poor fraction a three-dimensional fibrin matrix is created and filled the previously curetted fistulous tract. We describe the results in terms of demographic variables, outcomes, surgical procedure and complications. Endpoint was fistula closure by physical examination and endoanal ultrasound. Results Sixty seven percent of the patients were female, mean age was 43 (26y SD). The clinical characteristics of the patients are described in table 1. All of the patients had previous abscess drainage surgery. The most frequent fistula was mid-transsphincteric type. 66.7% had seton at the time of surgery and only one patient had no medical treatment at that time. The median follow-up was 30 months. The recurrence rate was 66.7% with no differences with in age, sex, comorbidity, type of fistula, fistula location, medical treatment and whether they had a seton implanted at the time of surgery. No patient presented complications. The median follow-up was 16 months. Conclusion PRGF sealing appears to be feasible, safe and a promising option in the treatment of PCD. Further studies should be carried out to determine the real use of PRGF in PCD.

Constitutive Active Mutant TIE2 Induces Enlarged Vascular Lumen Formation with Loss of Apico-basal Polarity and Pericyte Recruitment

Abnormalities in controlling key aspects of angiogenesis including vascular cell migration, lumen formation and vessel maturation are hallmarks of vascular anomalies including venous malformation (VM). Gain-of-function mutations in the tyrosine kinase receptor TIE2 can cause VM and induce a ligand-independent hyperactivation of TIE2. Despite these important findings, the TIE2-dependent mechanisms triggering enlarged vascular lesions are not well understood. Herein we studied TIE2 p.L914F, the most frequent mutation identified in VM patients. We report that endothelial cells harboring a TIE2-L914F mutation display abnormal cell migration due to a loss of front-rear polarity as demonstrated by a non-polarized Golgi apparatus. Utilizing a three-dimensional fibrin-matrix based model we show that TIE2-L914F mutant cells form enlarged lumens mimicking vascular lesions present in VM patients, independently of exogenous growth factors. Moreover, these abnormal vascular channels demonstrate a dysregulated expression pattern of apico-basal polarity markers Podocalyxin and Collagen IV. Furthermore, in this system we recapitulated another pathological feature of VM, the paucity of pericytes around ectatic veins. The presented data emphasize the value of this in vitro model as a powerful tool for the discovery of cellular and molecular signals contributing to abnormal vascular development and subsequent identification of novel therapeutic approaches.

The Upregulation of Integrin αDβ2 (CD11d/CD18) on Inflammatory Macrophages Promotes Macrophage Retention in Vascular Lesions and Development of Atherosclerosis.

Macrophage accumulation is a critical step during development of chronic inflammation, initiating progression of many devastating diseases. Leukocyte-specific integrin αDβ2 (CD11d/CD18) is dramatically upregulated on macrophages at inflammatory sites. Previously we found that CD11d overexpression on cell surfaces inhibits in vitro cell migration due to excessive adhesion. In this study, we have investigated how inflammation-mediated CD11d upregulation contributes to macrophage retention at inflammatory sites during atherogenesis. Atherosclerosis was evaluated in CD11d-/-/ApoE-/- mice after 16 wk on a Western diet. CD11d deficiency led to a marked reduction in lipid deposition in aortas and isolated macrophages. Macrophage numbers in aortic sinuses of CD11d-/- mice were reduced without affecting their apoptosis and proliferation. Adoptive transfer of fluorescently labeled wild-type and CD11d-/- monocytes into ApoE-/- mice demonstrated similar recruitment from circulation, but reduced accumulation of CD11d-/- macrophages within the aortas. Furthermore, CD11d expression was significantly upregulated on macrophages in atherosclerotic lesions and M1 macrophages in vitro. Interestingly, expression of the related ligand-sharing integrin CD11b was not altered. This difference defines their distinct roles in the regulation of macrophage migration. CD11d-deficient M1 macrophages demonstrated improved migration in a three-dimensional fibrin matrix and during resolution of peritoneal inflammation, whereas migration of CD11b-/- M1 macrophages was not affected. These results prove the contribution of high densities of CD11d to macrophage arrest during atherogenesis. Because high expression of CD11d was detected in several inflammation-dependent diseases, we suggest that CD11d/CD18 upregulation on proinflammatory macrophages may represent a common mechanism for macrophage retention at inflammatory sites, thereby promoting chronic inflammation and disease development.

Assessing angiogenic responses induced by primary human prostate stromal cells in a three-dimensional fibrin matrix assay.

Accurate modeling of angiogenesis in vitro is essential for guiding the preclinical development of novel anti-angiogenic agents and treatment strategies. The formation of new blood vessels is a multifactorial and multi-stage process dependent upon paracrine factors produced by stromal cells in the local microenvironment. Mesenchymal stem cells (MSCs) are multipotent cells in adults that can be recruited to sites of inflammation and tissue damage where they aid in wound healing through regenerative, trophic, and immunomodulatory properties. Primary stromal cultures derived from human bone marrow, normal prostate, or prostate cancer tissue are highly enriched in MSCs and stromal progenitors. Using conditioned media from these primary cultures, a robust pro-angiogenic response was observed in a physiologically-relevant three-dimensional fibrin matrix assay. To evaluate the utility of this assay, the allosteric HDAC4 inhibitor tasquinimod and the anti-VEGF monoclonal antibody bevacizumab were used as model compounds with distinct mechanisms of action. While both agents had a profound inhibitory effect on endothelial sprouting, only bevacizumab induced significant regression of established vessels. Additionally, the pro-angiogenic properties of MSCs derived from prostate cancer patients provides further evidence that selective targeting of this population may be of therapeutic benefit.

Quantitative Analysis of Cellular Traction Generation and Actomyosin Dynamics in a 3D Fibrin Matrix

Substantial progress has been made in characterizing the mechanisms of tension sensing, adhesion, and traction in cells migrating on two-dimensional (2D), rigid surfaces. However, there are inherent differences in cellular architecture, signaling, and behavior when cells are embedded in the soft and confined environment of the extracellular matrix. Here we present a quantitative analysis of the dynamics of canonical actomyosin cytoskeletal proteins in primary dermal fibroblasts (HFFs) grown in a three-dimensional (3D) fibrin matrix, and correlate these dynamics to protrusion formation and traction generation during migration through the fibrin network. We observe that fibroblasts adopt an elongated cell morphology with adhesive protrusions that cyclically strain the surrounding fibrin matrix. We use GFP-tagged myosin regulatory light chain (MRLC1) and time-lapse confocal imaging to track myosin dynamics as cells explore the fibrin matrix. We observe highly dynamic alterations in MRLC1 localization coupled to both the extension and retraction of cellular protrusions. During protrusion extension, fibrin strain is often oriented bidirectionally several microns behind the protrusion tip and is transmitted via paxillin-dependent adhesions. ROCK inhibition by Y-27632 causes fibroblasts to develop extended protrusions despite a pronounced reduction in matrix deformation and adhesion maturation as judged by a dramatic reduction in matrix-associated paxillin plaques. Ongoing work characterizes the contribution of cytoskeletal contractility in regulating protrusion and adhesion dynamics.

Effect of a dienogest for an experimental three-dimensional endometrial culture model for endometriosis

The pathogenesis of endometriosis remains poorly understood at least in part because early stages of the disease process are difficult to investigate. Previous studies have proposed a three-dimensional fibrin matrix culture model to study human endometriosis. We examined the ultrastructural features of the endometriosis in this model and assessed the effect of a progestin on endometrial outgrowth and apoptosis in this culture system. Endometrial explants were placed in three-dimensional fibrin matrix culture and treated with and without various concentrations of the progestin dienogest. By the second week, endometrial gland-like formation was established in outgrowths both attached to and at a distance from the explants. These cells formed a combination of clumps and tubular monolayers surrounding a central cavity. Electron microscopy demonstrated that these cells are polarized with microvilli on the apical surface, desmosome-like structures, and basement membrane; features consistent with glandular epithelial cells. Outgrowth of endometrial stromal cells and glandular formation was impaired in response to dienogest in a dose-dependent manner. Our study shows that the human endometrial explants cultured in three-dimensional fibrin matrix establish outgrowths that ultrastructurally resemble ectopic endometrial implants. This model may provide insight into the cellular processes leading to endometriosis formation and enables screening of therapeutic compounds.

Platelet rich plasma extract promotes angiogenesis through the angiopoietin1-Tie2 pathway

Development and regeneration of tissues and organs require precise coordination among endothelial, epithelial and mesenchymal morphogenesis. Angiogenesis plays key roles in normal development, wound healing, recovery from ischemic disease, and organ regeneration. It has been recognized that the combination of various angiogenic factors in an appropriate physiological ratio is critical for long-term functional blood vessel formation. Here we show that mouse soluble platelet-rich-plasma (PRP) extract, which includes abundant angiopoetin-1 (Ang1) and other angiogenic factors, stimulates endothelial cell growth, migration and differentiation in cultured human dermal microvascular endothelial cells in vitro and neonatal mouse retinal angiogenesis in vivo. Mouse platelet rich fibrin (PRF) matrix, the three-dimensional fibrin matrix that releases angiogenic factors with similar concentrations and proportions to the PRP extract, also recapitulates robust angiogenesis inside the matrix when implanted subcutaneously on the living mouse. Inhibition of Ang1-Tie2 signaling suppresses PRP extract-induced angiogenesis in vitro and angiogenic ability of the PRF matrix in vivo. Since human PRP extract and PRF matrix can be prepared from autologous peripheral blood, our findings may lead to the development of novel therapeutic interventions for various angiogenesis-related diseases as well as to the improvement of strategies for tissue engineering and organ regeneration.

Modulation of 3D Fibrin Matrix Stiffness by Intrinsic Fibrinogen–Thrombin Compositions and by Extrinsic Cellular Activity

Fibrin is a substance formed through catalytic conversion of coagulation constituents: fibrinogen and thrombin. The kinetics of the two constituents determines the structural properties of the fibrin architecture. We have shown previously that changing the fibrinogen and thrombin concentrations in the final three-dimensional (3D) fibrin matrix influenced cell proliferation and differentiation. In this study, we further examined the effect of changing fibrinogen and thrombin concentrations in the absence or presence of fibroblasts on the structural modulus or stiffness of 3D fibrin matrices. We have prepared fibroblast-free and fibroblast-embedded 3D fibrin matrices of different fibrinogen and thrombin formulations, and tested the stiffness of these constructs using standard mechanical testing assays. Results showed that there was a corresponding increase in stiffness with increasing thrombin and fibrinogen concentrations; the increase was more notable with fibrinogen and to a lesser degree with thrombin. The effect of fibroblasts on the stiffness of the fibrin construct was also examined. We have observed a small increase in the stiffness of the fibroblast-incorporated fibrin construct as they proliferated and exhibited spreading morphology. To our knowledge, this is the first comprehensive report detailing the relationship between fibrinogen and thrombin concentrations, cell proliferation, and stiffness in 3D fibrin matrices. The data obtained may lead to optimally design suitable bioscaffolds where we can control both cell proliferation and structural integrity for a variety of tissue engineering applications.

Blood outgrowth endothelial cells from cord blood and peripheral blood: angiogenesis-related characteristics in vitro.

Blood outgrowth endothelial cells (BOEC) are good candidates for vascular (re-) generating cell therapy. Although cord blood (CB) BOEC have been reported as more proliferative than peripheral blood (PB) BOEC, not much is known about their functional properties. We have studied the following determinants in BOEC expanded from CB and PB: endothelial phenotype, in vitro adhesion, migration, proliferation, and angiogenic tube forming capacity. Endothelial phenotype of BOEC was evaluated by fluorescence activated cell sorting (FACS) analysis and confirmed the presence of endothelial markers including CD31, CD105, CD144, CD146, KDR/VEGFR-2, Tie-2, and TNF-alpha-induced VCAM-1 and ICAM-1. Evaluation of cell proliferation revealed a higher basal proliferation of CB-BOEC, which increased after exposure to bFGF but not VEGF. The lower basal proliferation of PB-BOEC increased with VEGF or bFGF addition. Array analysis of angiogenic genes showed many comparable expressions in both BOEC, and a slightly more pronounced pro-angiogenic profile in CB-BOEC than PB-BOEC. Both BOEC were able to form tubular structures in a three-dimensional fibrin matrix. Tube formation in CB-BOEC was markedly induced by TNF-alpha only and inhibited by anti-urokinase antibodies. It was comparable to that induced by combined addition of TNF-alpha and VEGF or bFGF, while maximal tube formation in PB-BOEC required simultaneous exposure to TNF-alpha/VEGF or TNF-alpha/bFGF. The endothelial phenotype and characteristics for homing, adhesion, migration, inflammation, and angiogenic tube formation are almost equal for BOEC from CB and PB. A slightly more angiogenic phenotype favors CB-BOEC. However, addition of VEGF to PB-BOEC induces equal proliferation and tube formation.

Letrozole stimulates the growth of human endometrial explants cultured in three-dimensional fibrin matrix

To investigate the effects of an aromatase inhibitor, letrozole, on the growth of human endometrium in a three-dimensional fibrin matrix model of endometriosis. Experimental study of human endometrial biopsies in a three-dimensional fibrin matrix culture system. Academic research center. Eight normal women with benign gynecologic problems. Endometrial biopsy samples were washed, cut into small pieces, and placed between two layers of fibrin gel in the presence or absence of letrozole in the culture medium. Tissue changes were assessed by histological and immunohistochemical staining using an inverted microscope, image analysis, and a semiquantitative scoring system. Stromal and epithelial cell outgrowth into the fibrin matrix and angiogenesis comprising endothelial cell invasion of the matrix. Letrozole (0.1 micromol/L, 1 micromol/L, and 10 micromol/L) exerted a significant growth stimulation effect on endometrial tissue in this model. In contrast to our expectations, letrozole stimulated growth of normal human endometrium in an in vitro model of endometriosis. Normal endometrium may respond differently than endometriotic lesions to therapeutic agents. Our findings should be kept in mind when considering future research to explore new clinical treatments for endometriosis.

Differential gene expression analysis of tubule forming and non-tubule forming endothelial cells: CDC42GAP as a counter-regulator in tubule formation

The formation of new tubular structures from a quiescent endothelial lining is one of the hallmarks of sprouting angiogenesis. This process can be mimicked in vitro by inducing capillary-like tubular structures in a three-dimensional (3D) fibrin matrix. We aimed to analyze the differential mRNA expression in two phenotypically distinct cell populations from the same culture, namely in tubule-forming endothelial cells and monolayer endothelial cells not participating in tubule formation. A fibrin-rich 3D matrix derived from human plasma was used to facilitate tubule formation by human foreskin microvascular endothelial cells (hMVEC). After 7 days of stimulation with VEGF, bFGF, and TNF-alpha, the culture consisted of a monolayer and capillary-like sprouts that had grown into the fibrinous matrix. A method was developed to separate the monolayer and tubule-forming populations of hMVEC, keeping their cellular integrity intact to ensure mRNA extraction and cDNA production. Subsequent array analysis resulted in an inventory of differentially expressed genes that were associated with either tube-forming (angiogenic) or non-angiogenic capacity. Differential gene expression was verified by real-time PCR on the original RNA samples as well as on RNA obtained from laser-capture microdissected cross sections of monolayers and capillary structures in the 3D fibrinous matrix. The expression of CDC42GAP, an inhibitor of active-state small Rho GTPases, was reduced in tubular hMVEC. Overexpression of CDC42GAP in hMVEC attenuated endothelial tubule formation, while its suppression by siRNA slightly enhanced this process. Thus, CDC42GAP was identified as a counter-regulatory mediator for tubule formation.

Effects of negative pressure wound therapy on fibroblast viability, chemotactic signaling, and proliferation in a provisional wound (fibrin) matrix

Vacuum Assisted Closure brand Negative Pressure Wound Therapy (V.A.C. NPWT) has been shown to be an effective therapeutic option for the treatment of recalcitrant wounds; however, the mechanism of action at the cellular level remains to be elucidated. Here, we examined the effects of negative pressure wound therapy, manifolded with two different dressings, on fibroblast viability, chemotactic signaling, and proliferation in a fibrin clot matrix. Fibroblasts were grown in a three-dimensional fibrin matrix and were treated for 48 hours with either V.A.C. NPWT and GranuFoam Dressing, or with gauze under suction, or as static controls without negative pressure or dressings. Cells treated by gauze under suction showed significantly greater cell death and stimulated less migration and proliferation than static and V.A.C. NPWT-treated cells (p<0.05). Apoptosis was also significantly higher in gauze under suction than in static treatments. These results indicate that the dressing material has a significant effect on cell response following negative pressure wound therapy. The ability to support cell growth, stimulate chemotaxis, and proliferation without increasing apoptosis may provide an insight into the mechanisms of action of V.A.C. NPWT.

Adhesive and Proteolytic Phenotype of Migrating Endothelial Cells Induced by Thymosin β‐4

The early stages of angiogenesis are usually accompanied by the occurrence of vascular leakage, and the deposition of fibrin in extravascular spaces. Initially, the fibrin network acts as a sealing matrix, but later on also as a scaffolding for invading endothelial cells. This process is induced by angiogenic growth factors, particularly by vascular endothelial growth factor (VEGF). Angiogenesis involves proteolytic activities, in particular cell-bound urokinase/plasmin and matrix metalloproteinase (MMPs) activities that modulate the fibrin structure and affect adhesion and migration of endothelial cells. Recent data show that formation of new vessels may be stimulated by thymosin beta-4 (Tbeta-4), but it is still not clear whether Tbeta-4 alone is angiogenic or the angiogenic potential of Tbeta-4 is mediated by VEGF. In this report to further characterize Tbeta-4 angiogenic activity, we produced its mutants that were deprived of the N-terminal tetrapeptide AcSDKP (Tbeta-4((AcSDKPT/4A))), the actin-binding sequence KLKKTET (Tbeta-4((KLKKTET/7A))) and with the nuclear localization sequence damaged by a point mutation Lys16Ala (Tbeta-4((K16A))). Then we tested their activity to induce expression and release of MMPs as well as plasminogen activators inhibitor type-1 (PAI-1). We also analyzed their effect on migration and proliferation of endothelial cells in three-dimensional (3D) fibrin matrix as well as on their ability to stimulate the outgrowth of human endothelial cells in capillary-like tubular structures. Our data demonstrate that increased intracellular expression of Tbeta-4 and its mutants is necessary and sufficient to induce PAI-1 gene expression in endothelial cells. Similarly, they stimulate expression and release of MMP-1, -2, and -3. As evaluated by using specific inhibitors to these MMPs, they modified specifically the structure of fibrin and thus facilitated migration of endothelial cells. To sum up, our data show that the mechanism by which Tbeta-4 induced transition of endothelial cells from quiescent to proangiogenic phenotype is characterized by increased expression of PAI-1 and MMPs did not require the presence of the N-terminal sequence AcSDKP, and depended only partially on its ability to bind G-actin or to enter the nucleus.

Composite fibrin and collagen scaffold to enhance tissue regeneration and angiogenesis

The goal of this study is to determine optimal composition of fibrin matrix and to develop a smart composite fibrin and collagen scaffold for tissue regeneration and angiogenesis. Human dermal fibroblasts (HDFs) showed more rapid F-actin assembly and cell migration in a three-dimensional fibrin matrix consisting of a physiologic range of fibrinogen concentrations. Microvascular and cellular densities of the composite fibrin and collagen scaffold implanted into nude mice were significantly higher than for a porous collagen matrix without fibrin ( p < 0.01). Histomorphometrically, stromal cell ingrowths, CD31-positive microvasculatures, and deposition of ECM matrix were more evenly distributed in the composite scaffold. The results demonstrate that fibrin admixed with porous collagen sponges can be used as a smart template for tissue regeneration.

Mechanical stimulation induces morphological and phenotypic changes in bone marrow‐derived progenitor cells within a three‐dimensional fibrin matrix

One of the major limitations in tissue engineering is cell sourcing. Multipotent progenitor cells appear to have many promising features for that purpose. Mechanical stimulation is known to play an important role in determining cell phenotype. The aim of this work was to investigate the effects of cyclic stretch on rat bone marrow derived progenitor cell (BMPC) morphology and smooth muscle-directed differentiation within a three-dimensional fibrin matrix. BMPCs were suspended in a fibrin gel, pipetted into the trough of Flexcell Tissue-Train plates, and stimulated with 10% longitudinal cyclic stretch at 1 Hz for 6 days. Unconstrained (stress- and strain-free) and static anchored (constrained but not stretched) samples were used as controls. Stress filament area per cell was increased in the stretched samples compared to static anchored and free-float controls. Cells in the free float controls were randomly aligned, while they aligned parallel to the direction of the stress or strain in the other groups. Immunofluorescence suggested an increased expression of smooth muscle markers (smooth muscle alpha actin and h1-calponin) in both stretched and constrained control samples, but not in unconstrained controls. Qualitative assessment suggested that collagen production was increased in both mechanically stimulated samples. Proliferation was inhibited in stretched samples compared to the constrained controls. This work suggests an ability of rat BMPCs to differentiate toward a smooth-muscle-cell-like lineage when exposed to biomechanical stimulation in a three-dimensional model. The observation that the constrained samples induced changes in BMPCs suggests that stress alone may be stimulatory, but addition of cyclic stretch appears to augment the responses.

Expression of Glycodelin and Cyclooxygenase‐2 in Human Endometrial Tissue Following Three‐dimensional Culture

Our previous study showed that in vitro culture of human endometrial tissue in a three-dimensional (3D) fibrin matrix could mimic the early stages of endometriosis with invasion, gland and stroma formation and sprouting of new vessels. The objective of the present study was to evaluate the expression of glycodelin (Gd) and cyclooxygenase-2 (COX-2), two angiogenic factors, to further validate the 3D culture model of endometriosis. Human endometrial fragments were obtained from endometrial biopsies and placed in a 3D fibrin matrix culture. Immunohistochemistry with specific antibodies to Gd and COX-2 was used to examine endometrial epithelium and blood vessels, and 4, 6-diamidino-2-phenylindole staining was used for nuclear identification. Three-dimensional culture of human endometrial tissue in the fibrin matrix resulted in the proliferation of endometrial stromal cells, glandular epithelium and angiogenesis. Gd positive glandular epithelium was seen in 85% of wells with developing endometrial glands and COX-2 positive new vessels were seen in 80% of wells with angiogenesis-like structures after 4 weeks of culture. Our findings confirm that angiogenesis occurs following the culture of endometrial tissue in the 3D fibrin matrix, and suggests that Gd and COX-2 might play important roles in promoting neovascularization and cell proliferation in the establishment of endometriosis.

Effect of a statin on an in vitro model of endometriosis

To determine the inhibitory effect of a statin on angiogenesis in a three-dimensional (3-D) culture of human endometrial fragments in vitro. Angiogenesis has been proposed as an important mechanism in the pathogenesis of endometriosis, and statins (3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors) have been shown to have anti-inflammatory and anti-angiogenic activity. Experimental in vitro study of human endometrial biopsies and 3-D culture in fibrin matrix. Research laboratory at a university-affiliated infertility center. Forty-six normal ovulating women undergoing infertility treatment. Endometrial samples obtained from the fundus of the uterine cavity were minced, and the fragments were placed in a three-dimensional fibrin matrix culture system. Presence or absence of proliferation of stromal cells and invasion of the fibrin matrix, presence or absence of vessel sprouting, and immunohistochemical characterization of cellular components. During the 1st week of culture, invasion of stromal cells into the fibrin matrix occurred in the control group and in some wells outgrowths were observed. After 2 weeks, endometrial glands were observed in the outgrowths at a distance from the main tissue and were growing in conjunction with new vessel formation until the end of culture period. A concentration-dependent effect of lovostatin was seen on cell growth and angiogenesis in the experimental groups. In the presence of 5 and 10 microM of statin, angiogenesis was abolished, and cell proliferation was inhibited. In the presence of 1 microM of lovastatin, angiogenesis was reduced, but cell proliferation was not affected. The statins were shown to be effective in inhibiting the mechanisms of cell proliferation and angiogenesis in an experimental model for the development of endometriosis-like tissue.

Tissue Engineering of Injectable Muscle: Three-Dimensional Myoblast-Fibrin Injection in the Syngeneic Rat Animal Model

Surgical treatment of skeletal muscle loss resulting from trauma, tumor ablation, or inborn tissue defects is hampered by the scarcity of functional substitute tissue. By using techniques of tissue engineering, reconstitution of skeletal muscle defects might become a more viable option. However, it is necessary to develop an adequate, practical method for delivering myoblasts within a three-dimensional scaffold in vivo. The aim of this study was to create and evaluate a novel method for the transfer of myoblasts with clinically approved components within a three-dimensional matrix. The authors injected expanded primary male myoblasts into muscle defects in female syngeneic rats using a two-way syringe (Duploject) within a three-dimensional fibrin matrix. Detection and evaluation were performed using Y chromosome in situ hybridization, antidesmin immunostaining, and hematoxylin and eosin staining. To identify possible differences by means of integration, the injected myoblasts were compared with 7 days of precultivated constructs. Injected myoblasts showed increasing integration into host muscle fibers in a time-dependent manner, exclusively at the injection site. Antidesmin staining revealed a conserved myogenic phenotype of transplanted cells. The fibrin matrix resolved over a period of 12 weeks, with no indication of an inflammatory reaction. No significant difference in the number of detected Y chromosome-positive nuclei was found between the two transplantation groups. The presented technique of myoblast-fibrin injection indicates a clinical potential for reconstruction of skeletal muscle defects in vivo using a ready-to-use device in combination with tissue-engineering methods.

Isolated Pancreatic Islets in Three-Dimensional Matrices are Responsive to Stimulators and Inhibitors of Angiogenesis

The formation of a new microvasculature is essential for the long-term survival and function of the islet graft. In this study we examined endothelium of isolated pancreatic islets by stimulation with growth factors, different culture conditions, and genetic modification. We also inspected the effect of immunosuppressives used in human transplantation on angiogenesis. Isolated islets were embedded in a three-dimensional fibrin or Matrigel matrix. The effect of hyperglycemia, hypoxia, and the addition of VEGF and bFGF was investigated. We exposed islets from transgenic mice expressing the VEGF gene (RIP1VEGF-A) to high glucose (16.7 mmol/L) medium and tested the immunosuppressive agents rapamycin (100 ng/ml) and FK506 (100 ng/ml). To quantify angiogenesis the percentage of sprouting islets was determined. New endothelial capillary-like structures protruded from isolated pancreatic islets. Addition of VEGF to the islets and transgenic RIP-VEGF islets showed a two- to threefold increase of sprouting islets compared to control. Hypoxic culture conditions stimulated angiogenesis, resulting in a twofold increase of capillary sprouting. Rapamycin and FK506 proved to be potent inhibitors of angiogenesis in this system, because a decrease of sprouting islets of more than 20% by both agents was observed. Isolated pancreatic islets are capable of forming new capillary structures and are susceptible to pro- and antiangiogenic stimuli.