In vitro culture of chondrocytes in a novel thermoreversible gelation polymer scaffold containing growth factors.

In this study we examined the potential of a novel thermoreversible gelation polymer (TGP) to act as a 3-D hydrogel scaffold and deliver both chondrocytes and growth factors. Chondrocytes obtained from bovine articular cartilage were studied as a suspension in TGP chilled to 4 degrees C, in the presence or absence of the growth factors IGF-1 and/or TGF beta2. The cold cell/aqueous suspensions were injected into a cylindrical mold and cultured at 37 degrees C for up to 16 weeks. Specimens obtained at 12 and 16 weeks were semitranslucent and elastic. The matrices surrounding the chondrocytes were histologically positive to Safranin-O staining and type II collagen staining. The glycosaminoglycan and hydroxyproline contents in the specimens increased as a function of time and because of the presence of growth factors; those cultured with growth factors produced significantly more of these substances than those cultured without. We have concluded that TGP has potential as a scaffold material in the generation of tissue-engineered cartilage in vitro.

An experimental study of apico-aortic valved conduit (AAVC) for surgical treatment of aortic stenosis in dogs.

A new valved conduit was developed using a canine aortic valve. The bioprosthetic valve was fixed with glutaraldehyde and epoxy compound (Denacol-EX313/810). A vascular graft composed of ultra-fine polyester fiber (10 mm in diameter, 200 mm in length) was used. Four dogs underwent apico-aortic valved conduit (AAVC) implantation and aortic banding (bypass group, BG), while another 4 dogs underwent aortic banding without AAVC implantation (control group, CG). Cardiac catheterization and angiocardiography were performed for assessment of hemodynamics 2 weeks and 6 months after surgery. Left ventricular systolic pressure, left ventricular end-diastolic pressure and the left ventricular-aortic pressure gradient differed significantly (P<0.01) between the BG and CG dogs. Left ventricular angiocardiography showed patency of the valved conduit in all the BG dogs. Echocardiography was performed before and 2, 4 and 6 months after surgery, and showed that while pressure overload caused concentric myocardial hypertrophy in the CG dogs, the left ventricle dilated eccentrically in the BG dogs. Furthermore, relief of left ventricular pressure overload by AAVC was maintained.

Successful transportation of human corneal endothelial tissues without cool preservation in varying Indian tropical climatic conditions and in vitro cell expansion using a novel polymer.

BACKGROUND: Though the transplantation of human corneal endothelial tissue (CET) separated from cadaver cornea is in practice, its transportation has not been reported. We report the successful transportation of CET in varying Indian climatic conditions without cool preservation and the in vitro expansion of Human Corneal Endothelial Precursor Cells (HCEPCs) using a novel Thermo-reversible gelation polymer (TGP). MATERIALS AND METHODS: CET from cadaver corneas (n = 67), unsuitable for transplantation, were used. In phase I, CET was transported in Basal Culture Medium (Group I) and TGP (Group II) and in Phase II, in TGP cocktail alone, from three hospitals 250-2500 km away, to a central laboratory. The transportation time ranged from 6 h to 72 h and the outdoor temperature between 20°C and 41°C. On arrival, CET were processed, cells were expanded upto 30 days in basal culture medium (Group A) and TGP scaffold (Group B). Cell viability and morphology were documented and Reverse transcription polymerase chain reaction (RT-PCR) characterization undertaken. RESULTS: In Phase I, TGP yielded more viable cells (0.11 × 10(6) cells) than Group I (0.04 × 10(6) cells). In Phase II, the average cell count was 5.44 × 10(4) cells. During expansion, viability of HCEPCs spheres in TGP was maintained for a longer duration. The cells from both the groups tested positive for B-3 tubulin and negative for cytokeratins K3 and K12, thereby proving them to be HCEPCs. CONCLUSION: TGP preserves the CET during transportation without cool preservation and supports in vitro expansion, with a higher yield of HCEPCs, similar to that reported in clinical studies.

Ex vivo cultivation of corneal limbal epithelial cells in a thermoreversible polymer (Mebiol Gel) and their transplantation in rabbits: an animal model.

We evaluated the efficacy of autologous expanded corneal epithelial cell transplants derived from harvested limbal biopsy cultured on a thermoreversible polymer (Mebiol Gel) for the management of unilateral limbal stem cell deficiency (LSCD). Corneal limbal biopsies from 12 rabbits were cultured on a thermoreversible polymer Mebiol Gel at 37 degrees C. Cells were harvested from the dishes after 3 weeks by reducing temperature to 4 degrees C. Autologous transplantation was undertaken to reconstruct the experimentally induced limbal stem cell deficiency in the rabbit eyes. The corneas of both eyes of all rabbits were harvested later for molecular studies. Reparative surgery was a total success in seven rabbits, partial success in two, and failure in three eyes. Histology of the seven successful eyes showed the successful growth of the corneal epithelium. Immunohistochemistry and reverse transcriptase polymerase chain reaction showed the cornea phenotype and stem cell-associated markers in the limbus of the seven successful eyes, indicating the homing of these cells into limbus. In the three failure cases and in the two control rabbit eyes, used in the study, histology showed presence of goblet cells and vascularization in the stroma with abortive formation of corneal epithelium. Our results suggest that transplantation of autologous limbal epithelial cells grown in thermoreversible polymer Mebiol Gel may restore a nearly normal ocular epithelial surface in eyes with unilateral LSCD.

Endovascular treatment of experimental aneurysms using a combination of thermoreversible gelation polymer and protection devices: feasibility study.

OBJECTIVE: We developed a new liquid embolic agent, an aqueous solution of thermoreversible gelation polymer (TGP) for the treatment of cerebral aneurysms. This polymer solution has the unique characteristics that allow it to solidify at a specific temperature without solvent. We performed an experimental aneurysm embolization using this liquid embolic agent with and without different protective devices to evaluate its technical feasibility for the treatment of aneurysms. METHODS: Fourteen side-wall aneurysms were surgically constructed on 14 common carotid arteries of 7 swine. Embolizations were conducted in combination with balloon protection (balloon group, n = 4), microstent protection (stent group, n = 4), and microcoil and microstent protection (stent-coil group, n = 4). Two aneurysms were used as controls. One control aneurysm was not embolized, and the other received control stent placement only. Angiographic follow-up was performed on day 14 and was followed by histopathological evaluation. RESULTS: Successful TGP solution delivery was conducted in all cases. Complete aneurysm occlusion was achieved in all cases without TGP migration. Follow-up angiograms demonstrated complete occlusion in the stent and stent-coil groups. A small recurrence was observed in the balloon group. Histopathological findings demonstrated neoendothelialization across the necks of the aneurysms. CONCLUSION: Experimental aneurysms were safely embolized using TGP. Further modifications related to mechanical stability and long-term safety evaluation results are necessary before clinical application.

Novel drug delivery system using thermoreversible gelation polymer for malignant glioma.

Many approaches to local tumor treatment have been reported and their efficacy demonstrated in patients with malignant glioma. We studied thermoreversible gelation polymer (TGP) as a novel drug delivery system (DDS) for treating this type of tumor. TGP exhibits sol-gel transition i.e., is water-soluble in the sol phase below the chosen sol-gel transiting temperature and water-insoluble in the gel phase above this temperature. We conjugated doxorubicin with TGP to prepare doxorubicin-TGP (DXR-TGP), then studied the kinetics of doxorubicin release from TGP and the antitumor activity of DXR-TGP in vitro and in vivo. The diffusive speed of doxorubicin from TGP was 9.4x10(-7) cm(2)/s and doxorubicin was reliably released from TGP. DXR-TGP showed antitumor activity against the human glioma cell lines T98G and U87MG and in a subcutaneous tumor model in nude mice. Pathologically, detection of the proliferation marker Ki-67 was considerably lower in the DXR-TGP group than in the control group (30-40% vs. 60-70%, respectively). This is to the best of our knowledge the first report of TGP as a novel drug delivery system, and further we provide evidence that TGP exhibits potential for use as a novel DDS for malignant glioma.

Separation and recovery of DNA fragments by electrophoresis through a thermoreversible hydrogel composed of poly(ethylene oxide) and poly(propylene oxide).

We have synthesized and characterized a thermoreversible hydrogel of multiplied block copolymers, composed of poly(ethylene oxide) and poly(propylene oxide), for DNA electrophoresis. The aqueous solution of block copolymers turned into a hydrogel upon heating at temperatures above 10-11 degrees C, whereas it reverted into a solution upon cooling. Linear double-stranded DNA molecules migrated through the gel matrices at a rate that was inversely proportional to the logarithm of the DNA length. The hydrogel is most effective for separating DNA fragments in the 10- to 2000-bp range. The resolving range lay in-between the effective ranges of polyacrylamide and agarose gel electrophoreses of DNA. The gel slices containing DNA fragments were liquefied by cooling on ice, and the DNA was precipitated with ethanol. No contaminants that inhibit enzymatic reactions were found in the DNA recovered from the hydrogel. Plasmid DNA recovered from the hydrogel was recircularized with T4 DNA ligase and yielded highly efficient Escherichia coli transformation. Therefore, thermoreversible gel electrophoresis will be a useful method for DNA separation and isolation in recombinant DNA technology.

Gene expression profile of human mesenchymal stem cells during osteogenesis in three-dimensional thermoreversible gelation polymer.

This study attempted to characterize the ability of thermoreversible gelation polymer (TGP) to induce differentiation of human mesenchymal stem cells (hMSC) into osteoblasts. Using a long oligo microarray system consisting of 3760 genes, we compared the expression profiles of the cells in 2-dimensional (2D) culture, 3D culture in collagen gel, and 3D culture in TGP with or without osteogenic induction. Compared to 2D culture, the gene expression profile of hMSC showed almost the same pattern in TGP without osteogenic induction, but 72% of genes (2701/3760) were up-regulated in collagen gel. With osteogenic induction, hMSC showed higher ALP activity and osteocalcin production in TGP as compared to 2D culture. Moreover, up-regulation and down-regulation of osteogenic genes were augmented in 3D culture in TGP as compared to 2D culture. As TGP is chemically synthesized and completely free from pathogen such as prion in bovine spongiform encephalopathy, these results suggest that TGP could be applied clinically to induce osteogenic differentiation of hMSC.