In vivo evaluation of scaffolds compatible for colonoid engraftments onto injured mouse colon epithelium.

Colon organoids (colonoids) are known to be similar to colon tissue in structure and function, which makes them useful in the treatment of intestinal de-epithelialized disease. Matrigel, which is used as a transplantation scaffold for colonoids, cannot be used in clinical applications because of its undefined composition and tumorigenicity. This study identifies clinically available scaffolds that are effective for colonoid transplantation in damaged intestinal mucosa. The colon crypt was isolated and cultured from C57BL/6-Tg[CAG enhanced green fluorescent protein (EGFP)131Osb/LeySopJ mice into EGFP + colonoids and subsequently transplanted into the EDTA colitis mouse model using gelatin, collagen, or fibrin glue scaffolds. To identify scaffolds suitable for colonoid engraftment in injured colon mucosa, the success rates of transplantation and secondary EGFP colonoid formation were measured, and the scaffolds' mediated toxicity in vitro and in vivo was observed in recipient mice. When colonoids were transplanted with gelatin, collagen, and fibrin glue into the EDTA colitis mouse model, all groups were found to be successfully engrafted. Fibrin glue, especially, showed significant increase in the engrafted area compared with Matrigel after 4 wk. The scaffolds used in the study did not induce colonic toxicity after transplantation into the recipients' colons and were thus deemed safe when locally administrated. This study suggests new methods for and provides evidence of the safety and utility of the clinical application of colonoid-based therapeutics. Furthermore, the methods introduced in this study will be helpful in developing cell treatment using the esophagus or a stomach organoid for various digestive-system diseases.-Jee, J., Jeong, S. Y., Kim, H. K., Choi, S. Y., Jeong, S., Lee, J., Ko, J. S., Kim, M. S., Kwon, M.-S., Yoo, J. In vivo evaluation of scaffolds compatible for colonoid engraftments onto injured mouse colon epithelium.

Fabrication and characterization of 3D microtubular collagen scaffolds for peripheral nerve repair.

Understanding the structure-function relationship in biomaterial constructs is critical in optimizing biological outcomes. For ensheathed structures such as peripheral nerve, engineering implantable tissue substitutes has been challenging. This is due to a unique geometry of thin-walled microtube arrays composed mostly of basement membrane. In this work, we propose a sacrificial templating method to create Matrigel scaffolds that resemble endogenous peripheral nerve. These paralleled microtube constructs possess high void space and membrane-like walls. Additionally, we investigated the effect of chemical crosslinking in altering the physical, mechanical, and biologic properties of Matrigel. Results show that both glutaraldehyde and genipin increased the modulus and failure stress of Matrigel while also improving degradation resistance. However, glutaraldehyde crosslinking induced some cytotoxicity whereas genipin showed good biocompatibility. PC-12 cells, Schwann cells, and primary chick dorsal root ganglia cultured onto microtube scaffolds demonstrated viability up to 10 days. Strong cellular alignment along the channels was observed in Schwann cells whereas neurite outgrowth in primary chick dorsal root ganglia was also biased along the major axis of the microtubes. This suggests that the microtubes may mediate cell orientation and axon pathfinding. This proof of concept study provides a tunable workflow that may be adapted to other collagen types.

Induction of Angiogenesis by Matrigel Coating of VEGF-Loaded PEG/PCL-Based Hydrogel Scaffolds for hBMSC Transplantation.

hBMSCs are multipotent cells that are useful for tissue regeneration to treat degenerative diseases and others for their differentiation ability into chondrocytes, osteoblasts, adipocytes, hepatocytes and neuronal cells. In this study, biodegradable elastic hydrogels consisting of hydrophilic poly(ethylene glycol) (PEG) and hydrophobic poly(ε-caprolactone) (PCL) scaffolds were evaluated for tissue engineering because of its biocompatibility and the ability to control the release of bioactive peptides. The primary cultured cells from human bone marrow are confirmed as hBMSC by immunohistochemical analysis. Mesenchymal stem cell markers (collagen type I, fibronectin, CD54, integrin1ß, and Hu protein) were shown to be positive, while hematopoietic stem cell markers (CD14 and CD45) were shown to be negative. Three different hydrogel scaffolds with different block compositions (PEG:PCL=6:14 and 14:6 by weight) were fabricated using the salt leaching method. The hBMSCs were expanded, seeded on the scaffolds, and cultured up to 8 days under static conditions in Iscove's Modified Dulbecco's Media (IMDM). The growth of MSCs cultured on the hydrogel with PEG/PCL= 6/14 was faster than that of the others. In addition, the morphology of MSCs seemed to be normal and no cytotoxicity was found. The coating of the vascular endothelial growth factor (VEGF) containing scaffold with Matrigel slowed down the release of VEGF in vitro and promoted the angiogenesis when transplanted into BALB/c nude mice. These results suggest that hBMSCs can be supported by a biode gradable hydrogel scaffold for effective cell growth, and enhance the angiogenesis by Matrigel coating.

Inhibition of angiogenesis by ß-galactosylceramidase deficiency in globoid cell leukodystrophy.

Globoid cell leukodystrophy (Krabbe disease) is a neurological disorder of infants caused by genetic deficiency of the lysosomal enzyme ß-galactosylceramidase leading to accumulation of the neurotoxic metabolite 1-ß-d-galactosylsphingosine (psychosine) in the central nervous system. Angiogenesis plays a pivotal role in the physiology and pathology of the brain. Here, we demonstrate that psychosine has anti-angiogenic properties by causing the disassembling of endothelial cell actin structures at micromolar concentrations as found in the brain of patients with globoid cell leukodystrophy. Accordingly, significant alterations of microvascular endothelium were observed in the post-natal brain of twitcher mice, an authentic model of globoid cell leukodystrophy. Also, twitcher endothelium showed a progressively reduced capacity to respond to pro-angiogenic factors, defect that was corrected after transduction with a lentiviral vector harbouring the murine ß-galactosylceramidase complementary DNA. Finally, RNA interference-mediated ß-galactosylceramidase gene silencing causes psychosine accumulation in human endothelial cells and hampers their mitogenic and motogenic response to vascular endothelial growth factor. Accordingly, significant alterations were observed in human microvasculature from brain biopsy of a globoid cell leukodystrophy case. Together these data demonstrate that ß-galactosylceramidase deficiency induces significant alterations in endothelial neovascular responses that may contribute to central nervous system and systemic damages that occur in globoid cell leukodystrophy.

Study in vivo intraocular biocompatibility of in situ gelation hydrogels: poly(2-ethyl oxazoline)-block-poly(ε-caprolactone)-block-poly(2-ethyl oxazoline) copolymer, matrigel and pluronic F127.

The long term in vivo biocompatibility is an essential feature for the design and development of sustained drug release carriers. In the recent intraocular drug delivery studies, hydrogels were suggested as sustained release carriers. The biocompatibility test for these hydrogels, however, was commonly performed only through in vitro cell culture examination, which is insufficient before the clinical applications. We compared three thermosensitive hydrogels that have been suggested as the carriers for drugs by their gel-solution phase-change properties. A new block terpolymer (PEOz-PCL-PEOz, ECE) and two commercial products (Matrigel® and Pluronic F127) were studied. The results demonstrated that the ocular media remained translucent for ECE and Pluronic F127 in the first 2 weeks, but cataract formation for Matrigel occurred in 2 weeks and for Pluronic F127 in 1 month, while turbid media was observed for both Matrigel and Pluronic F127 in 2 months. The electrophysiology examinations showed significant neuroretinal toxicity of Matrigel and Pluronic F127 but good biocompatibility of ECE. The neuroretinal toxicity of Matrigel and Pluronic F127 and superior biocompatibility of ECE hydrogel suggests ECE as more appropriate biomaterial for use in research and potentially in intraocular application.

A subretinal matrigel rat choroidal neovascularization (CNV) model and inhibition of CNV and associated inflammation and fibrosis by VEGF trap.

PURPOSE: The exudative, or the wet form of age-related macular degeneration (AMD) is characterized by choroidal neovascularization (CNV). A subretinal Matrigel (BD Biosciences, Bedford MA) model of CNV is described here, along with the effects of vascular endothelial growth factor (VEGF) neutralization on the development of CNV and associated inflammation and fibrosis. METHODS: CNV was induced in adult Sprague-Dawley rats by subretinal injection of Matrigel. CNV growth and associated leukocyte infiltration and collagen deposition were examined. VEGF Trap (Regeneron Pharmaceuticals, Tarrytown, NY), a recombinant protein that comprises portions of the extracellular domains of VEGF receptors 1 and 2 and that binds all isoforms of VEGF-A as well as placental growth factor with high affinity, was administered subcutaneously. RESULTS: Initiation of CNV was detected 4 days after Matrigel injection and then increased progressively in size. Systemic administration of VEGF Trap beginning on day 2 and 6 completely prevented development of CNV. When CNV was allowed to develop for 10 days before treatment was initiated, VEGF Trap not only prevented its further progression, but also induced substantial regression of existing lesions. In addition, VEGF Trap treatment reduced the total lesion volume and largely prevented the progressive leukocyte infiltration and fibrosis associated with CNV. CONCLUSIONS: The subretinal Matrigel CNV model provides a convenient tool for the study of the diverse components of complex CNV lesions. The data not only confirm the critical roles of VEGF in the development and maintenance of CNV, but further demonstrate that VEGF and other VEGF receptor 1 ligands promote CNV-associated inflammation and fibrosis.

Exacerbation of retinal degeneration and choroidal neovascularization induced by subretinal injection of Matrigel in CCL2/MCP-1-deficient mice.

This study presents a mouse model for human age-related macular degeneration (AMD) as characterized by subretinal deposit and choroidal neovascularization. Matrigel, a basement membrane extract, solidifies after implantation in tissue and can stimulate local angiogenesis. This study demonstrates the induction of neovascularization and focal retinal degeneration following subretinal Matrigel injection in mice. In senescent mice, the normal functioning of CC chemokine CCL2/MCP-1 and its receptor CCR2 confers protection against age-related retinal degeneration, a disease that shares many similar features with human AMD. Our data shows that CCL2-deficient mice develop more severe disease as compared to the wild-type controls. These findings suggest that Matrigel subretinal injection could be used to generate AMD-like pathological changes. The data support the previously proposed role of CCL2 in AMD pathogenesis.

Angiocidal effect of Cyclosporin A: a new therapeutic approach for pathogenic angiogenesis.

AIM: Angiogenesis is essential in the development of several disorders such as cancer, arthritis, and autoimmune diseases. Several agents prevent angiogenesis but only a few destroy established angiogenesis. In this study we tested whether local or systemic administration of Cyclosporin A (CyA) would inhibit as well as destroy established angiogenesis in an in vivo assay of angiogenesis. METHODS: We utilized an in vivo assay of angiogenesis in which an angiogenic mixture of Matrigel, FGF, VEGF, and heparin was injected subcutaneously into mice. Angiogenesis in the subcutaneous plugs was quantified by ANOVA. CyA or the vehicle for CyA was administered to the experimental or the control groups by three routes: by addition to the angiogenic mixture, by local injection into the angiogenic plug at various time points or by systemic administration at high doses. Angiogenesis was quantified by pointing method and expressed as an angiogenic index (AI). RESULTS: In control animals the subcutaneous plug of Matrigel with the angiogenic mixture revealed exuberant angiogenesis at day 4 and day 7. This angiogenesis was completely inhibited when CyA was included in the angiogenic mixture; the vehicle for CyA had no such effect. Angiogenesis that had progressed was found to regress after local subcutaneous injection of CyA at day 4 and 7. Similar regression of angiogenesis was noted when CyA was administered systemically after allowing angiogenesis to proceed for 4 days. CONCLUSIONS: Our experiments strongly suggest that CyA is both angiocidal and angiostatic in vivo. These results provide a basis for future therapy directed against established angiogenesis in malignancies and autoimmune diseases.

Laminin-induced autoimmune myositis in rats.

The present study aimed to examine if immunization with laminin causes myositis in rats and whether the pathologic findings mirror human polymyositis and dermatomyositis. Rats were immunized with an emulsion of laminin and complete Freund's adjuvant. As a result, muscle fiber necrosis with infiltrating macrophages was frequently observed and mononuclear cells were observed in the endomysium. These mononuclear cells were composed of CD4+ cells, CD8+ T cells, and macrophages. CD4+ cells and CD8+ T cells were mainly located in the endomysium, whereas a large number of macrophages were located in the endomysium and infiltrating muscle fibers. A small number of B cells, detected by immunohistochemical staining, were mainly located in the perimysium. The nonnecrotic muscle fiber to which CD4+ T cells, CD8+ T cells, and perforin+ cells adhered was negative for antimerosin and antidystrophin antibodies. Muscle fiber necrosis in rats immunized with laminin may occur after denaturation of basement membrane proteins. In conclusion, the immunization with laminin induces moderate to severe myositis. We suggest that laminin may be an important antigen for connective tissue diseases such as polymyositis and dermatomyositis.

New human oral squamous carcinoma cell line and its tumorigenic subline producing granulocyte colony-stimulating factor.

A new human carcinoma cell line, MISK81-5, was established from a metastatic lymph node of oral squamous cell carcinoma. Immunocytochemical and ultrastructural observations revealed an obvious epithelial origin of the cell line. Chromosome analysis revealed a hypertriploid karyotype with numerical and structural anomalies. MISK81-5 cells could form a tumor mass in the subcutaneous tissue of recipient BALB/c athymic mice only when coinjected with Matrigel. A stem cell assay revealed that conditioned medium (CM) of MISK81-5 contained granulocyte colony-stimulating factor (G-CSF) or interleukin-6 activity. Quantitation by ELISA disclosed a higher concentration of G-CSF in the CM of MISK81-5 than in the CM of other squamous and gastric carcinoma cell lines. The sMISK, that was derived from MISK81-5 as a subpopulation of the cell line having higher tumorigenicity, also showed a similar hematopoietic stimulating activity to that of MISK81-5. These characteristics of the MISK81-5 cell line and its subpopulation, sMISK will be useful for studying the biological behavior of oral squamous cell carcinomas and its relation to hematopoietic stimulating factors.

Stimulation of angiogenesis as an explanation of Matrigel-enhanced tumorigenicity.

Matrigel, a reconstituted extract of basement membrane, enhances the growth of different human cancer cell lines when transplanted into nude mice. Here that stimulation was confirmed in the BALB/c murine mammary-tumor cell line M3MC, as well as in human colon (SW948) and mammary (MDA-MB-468) carcinoma cell lines transplanted in nude and SCID mice, respectively. Subcutaneous and intra-mammary fat-pad inoculations of Matrigel alone generated an angiogenic response which was macroscopically evident by day 9. Histological analysis of the local host reaction occurring at the site of injection revealed an early peripheral fibroblast response, followed by mononuclear cell infiltration, solid and hollow fibroblast cords projections from the edge to the center of the Matrigel plug, and finally capillary ingrowths. Conditioned media obtained from the gels generated in vivo, acted as very strong chemoattractants for mouse lung capillary endothelial cells, stimulating their motility between 38 and 82 times with respect to the control. Our results suggest an important role of host cells recruited by Matrigel, which could favor angiogenesis of the area and thus facilitate the growth of tumor cells co-inoculated with the basement membrane extract.

A screening method using tissue culture for evaluation of potential retinal adhesives.

Several adhesives have been tested for their potentially toxic effects on embryonic retinal tissue. The authors have characterized the effects of the adhesives on neurofilament extension and also on surgical-wound "re-knitting." While none of the adhesives in the sample (including those in current surgical use) seem to be ideal, the model advanced has application for the continuing development of better 'bio-adhesives'. The most immediate application is within the field of vitreoretinal surgery in situations where conventional procedures currently seem inadequate.