Tasima Haque

In vitro study of alginate-chitosan microcapsules : an alternative to liver cell transplants for the treatment of liver failure

The application of alginate–chitosan (AC) microcapsules to liver cell transplantation has not been previously investigated. In the current in vitro study, we have investigated the potential of AC microcapsules for the encapsulation of liver cells and show that the AC membrane supports the survival, proliferation and protein secretion by entrapped hepatocytes. The AC membrane provides cell immuno-isolation and has the potential for cell cryopreservation. The AC microcapsule has several advantages compared to more widely used alginate–poly-L-lysine (APA) microcapsules for the application of cell therapy.

Characterizing the BMP pathway in a wild type mouse model of distraction osteogenesis.

Distraction osteogenesis (DO) is a well established surgical technique for limb lengthening and replacement of bone loss due to trauma, infection or malignancies. Although the technique is widely used, one of its limitations is the long period of time required for the newly formed bone to consolidate. We have previously shown that exogenous application of bone morphogenetic proteins (BMPs) can increase bone formation during DO, however, exogenous BMPs have many drawbacks. An alternative method for accelerating the rate of bone formation may be to modulate the intrinsic BMP signaling pathway. The aim of the current study was to analyze the expression of various genes involved in the BMP pathway at various time periods during DO in order to identify potential targets for therapeutic manipulation. DO was applied to the right tibia of 80 adult wild type mice. Distraction began after a latency period of 5 days at a rate of 0.2 mm/12 h for 2 weeks. Mice were sacrificed in groups of 12 at the following times post surgery: day 5 (latency), days 11 and 17 (distraction) and days 34 and 51 (consolidation). Specimens were examined using radiology, microCT, histology, RT(2)PCR, immunohistochemistry and Western analysis. Genes involved in the BMP pathway including the BMP ligands, receptors, antagonists and downstream effectors were examined. A significant upregulation of BMPs 2, 4 and 6 was observed using both PCR and immunohistochemistry during the distraction phase. The expression of BMP7 remained constant throughout the distraction and consolidation process. Surprisingly, the only receptors which were upregulated significantly were the Activin Receptor Type 1 (ActR1) during distraction and Activin Receptor Type 2b (ActR2b) during consolidation. Most interestingly, simultaneously with the ligands, an increase in the expression of the antagonists, Noggin, Chordin, Inhibin and BMP3 was observed. This study provides a clearer understanding of expression patterns during DO, which is a valuable resource for finding therapeutic options to stimulate bone formation. The results suggest that blocking BMP inhibitors may be a possible method for increasing the function of intrinsic growth factors involved in bone regeneration.

Preparation and characterization of novel polymeric microcapsules for live cell encapsulation and therapy.

This article describes the preparation and in vitro characterization of novel genipin cross-linked alginate-chitosan (GCAC) microcapsules that have potential for live cell therapy applications. This microcapsule system, consisting of an alginate core with a covalently cross-linked chitosan membrane, was formed via ionotropic gelation between calcium ions and alginate, followed by chitosan coating by polyelectrolyte complexation and covalent cross-linking of chitosan by naturally derived genipin. Results showed that, using this design concept and the three-step procedure, spherical GCAC microcapsules with improved membrane strength, suppressed capsular swelling, and suitable permeability can be prepared. The suitability of this novel membrane formulation for live cell encapsulation was evaluated, using bacterial Lactobacillus plantarum 80 (pCBH1) (LP80) and mammalian HepG2 as model cells. Results showed that capsular integrity and bacterial cell viability were sustained 6 mo postencapsulation, suggesting the feasibility of using this microcapsule formulation for lives bacterial cell encapsulation. The metabolic activity of the encapsulated HepG2 was also investigated. Results suggested the potential capacity of this GCAC microcapsule in cell therapy and the control of cell signaling; however, further research is required.

Immunohistochemical localization of bone morphogenetic protein-signaling Smads during long-bone distraction osteogenesis.

In this study we investigated the expression of bone morphogenetic protein (BMP)-signaling Smads in distraction osteogenesis (DO). Osteotomy of the right tibia was performed in 14 skeletally mature white New Zealand male rabbits. Lengthening was started 1 week later at a rate of 0.5 mm/12 hr and was maintained for 3 weeks. Expression of Smad proteins 1, 4, 5, 6, 7, and 8 and Smad ubiquitin regulatory factors (Smurfs) 1 and 2 was evaluated in the distracted zone using immunohistochemistry. Expression of receptor-regulated Smads (R-Smads) 1, 5, and 8 showed a significant increase during the distraction phase, followed by a gradual decrease during the consolidation phase. Smad 4 showed significant expression during both distraction and the beginning of the consolidation phase. Smad 6 and Smad 7 were highly expressed during the consolidation phase. Staining for both Smurfs 1 and 2 was maximal at the end of the distraction period. Staining for all proteins was most intense in chondrocyte and fibroblast-like cells. Expression pattern of R-Smads correlated with our previously reported expression pattern of BMPs 2, 4, and 7 and their receptors. These results therefore suggest a role for the whole BMP signaling pathway including the Smad proteins in DO.

Early injection of OP-1 during distraction osteogenesis accelerates new bone formation in rabbits

Distraction osteogenesis (DO) is a surgical technique for generating new bone by applying controlled distraction of two bony segments post osteotomy. A limitation of the technique is the long time required for the new bone to consolidate. We investigated the effect of injecting osteogenic protein 1 (OP-1) at the beginning of distraction in a rabbit model of DO. Regenerate bone was evaluated using radiology, densitometry, micro-computed tomography (microCT) and histomorphometry. Immunohistochemsitry was used to evaluate changes in expression of various ligands, growth factors and receptors following OP-1 treatment. Compared to the control, a two-fold increase in bone volume was apparent for treated groups at 3 weeks post injection. An upregulation of almost all of the 41 genes examined was observed. Results suggested that applying OP-1 early during distraction can accelerate bone formation by the activation of numerous pathways. This study provides further insights on strategies to improve bone regeneration rate in DO.

In-vitro analysis of APA microcapsules for oral delivery of live bacterial cells

Oral administration of microcapsules containing live bacterial cells has potential as an alternative therapy for several diseases. This article evaluates the suitability of the alginate-poly-L-lysine-alginate (APA) microcapsules for oral delivery of live bacterial cells, in-vitro, using a dynamic simulated human gastro-intestinal (GI) model. Results showed that the APA microcapsules were morphologically stable in the simulated stomach conditions, but did not retain their structural integrity after a 3-day exposure in simulated human GI media. The microbial populations of the tested bacterial cells and the activities of the tested enzymes in the simulated human GI suspension were not substantially altered by the presence of the APA microcapsules, suggesting that there were no significant adverse effects of oral administration of the APA microcapsules on the flora of the human gastrointestinal tract. When the APA microcapsules containing Lactobacillus plantarum 80 (LP80) were challenged in the simulated gastric medium (pH = 2.0), 80.0% of the encapsulated cells remained viable after a 5-min incubation; however, the viability decreased considerably (8.3%) after 15 min and dropped to 2.6% after 30 min and lower than 0.2% after 60 min, indicating the limitations of the currently obtainable APA membrane for oral delivery of live bacteria. Further in-vivo studies are required before conclusions can be made concerning the inadequacy of APA microcapsules for oral delivery of live bacterial cells.

A new method for targeted drug delivery using polymeric microcapsules: Implications for treatment of Crohn's disease

Recent research and clinical evidence suggest that thalidomide could potentially be used to treat inflammation associated with Crohns disease. However, systemic side effects associated with large doses of this drug have limited its widespread use. Treatment, with thalidomide would prove more efficacious if the drug could be delivered directly to target areas in the gut, thereby reducing systemic circulation. Microcapsule encapsulation could enable direct delivery of the drug. To assess the latter, we designed and tested drug-targeting release characteristics of alginate-poly-l-lysine-alginate (APA) microcapsules in simulated gastrointestinal environments. The results show that APA capsules enabled delivery of thalidomide in the middle and distal portions of the small intestine. We also compared the APA membrane formulation with an earlier designed alginate chitosan (AC) membrane thalidomide formulation. The results show that both APA and AC capsules allow for successful delivery of thalidomide in the gut and could prove beneficial in the treatment of Crohns disease. However, further research is required.

Preparation and in vitro analysis of microcapsule thalidomide formulation for targeted suppression of TNF-α

Recent studies have implicated the cytokine tumor necrosis factor-alpha (TNF-α) in the inflammation associated with Crohns disease (CD). Thalidomide has been shown to decrease this inflammation by the suppression of TNF-α secretion. However, side effects associated with thalidomide have precluded its widespread usage. In the present study we investigated the efficacy of a “targeted delivery approach” for thalidomide at the site of inflammation. We observed that alginate-poly-l-lysine-alginate (APA) polymer-based microcapsule formulations that encapsulate thalidomide could be designed. These capsules could be delivered at target sites where they almost entirely suppress TNF-α secretion in lipopolysaccharide activated RAW 264.7 macrophage cells in vitro. These findings indicate that targeted delivery of thalidomide using APA capsules could facilitate its usage in reducing the inflammation associated with chronic conditions such as Crohns disease and ulcerative colitis.

Results and complications of a surgical technique for correction of coxa vara in children with osteopenic bones.

Background: Surgical correction of coxa vara in children with osteopenic bone diseases could be very challenging. In this study, we describe a modified surgical technique for the correction of coxa vara in children with bone fragility. We also report the results and complications of this technique in 16 children (21 coxa vara) with osteopenic bone diseases. Methods: Charts, clinic notes, and radiology images of 16 patients (21 hips) who had a proximal femoral osteotomy for the treatment of coxa vara in osteogenesis imperfecta (18 hips) and fibrous dysplasia (3 hips) in our institution between 1996 and 2005 were reviewed. The modified surgery involved an intertrochanteric osteotomy and the use of Kirshner wires and intramedullary rods. Neck-shaft angle, Hilgenreiner-epiphyseal angle, and head-shaft angle were assessed at preoperative, postoperative, and final follow-up. Results: The average age at surgery was 8.3 years (range, 3.3-15.8 years). The average correction of the neck-shaft angle was from 84.6 to 114.4 degrees, and for the Hilgenreiner-epiphyseal angle, it was from 67.7 to 42 degrees at final follow-up. All osteotomies were healed at 3 months postoperatively. The mean follow-up was 4.29 years (range, 1.70-8.12 years). The average improvement in abduction and external rotation of the hips was 14 and 15 degrees, respectively. There were no cases of infection and 2 cases (12%) of implant-related complications. One patient with fibrous dysplasia needed replacement of the intramedullary rod and additional distal femoral osteotomy 1 week postsurgery. In another patient, the intramedullary rod had migrated proximally, which was corrected 5 months postsurgery by advancing the rod distally. Conclusion: Results suggest that this surgical technique provides satisfactory correction of coxa vara in children with osteopenic bone diseases. Clinical Relevance: The described surgical technique used to correct coxa vara is reproducible and safe and has few complications. Furthermore, the size of the implants used allows surgery to be performed in very young children.

OP-1 injection increases VEGF expression but not angiogenesis in a rabbit model of distraction osteogenesis

We have previously shown that a single injection of rhBMP-7 (OP-1) applied to the regenerate early during distraction accelerates bone consolidation in a rabbit model of distraction osteogenesis. In the present study, we hypothesised that the injection of OP-1 improves bone consolidation by increasing blood flow to the distracted site. Blood flow into the regenerate of a rabbit model was measured and vascular endothelial growth factor (VEGF) expression was tested using semi-quantitative PCR. Immunohistochemistry was used for assessing the temporal and spatial expression of platelet endothelial cell adhesion molecule (PECAM), VEGF and its receptors following OP-1 injection. We observed a higher expression of VEGF and its receptors in the regenerate with OP-1 treatment. However, there was no difference in the increase in bone blood flow nor PECAM expression between the treated and control groups of animals. Interestingly, the increased expression of VEGF and its receptors was associated with chondrocyte and fibroblast-like cells, but not with endothelial cells. These results suggest that accelerated ossification by OP-1 may depend on a non-vascular mechanism, possibly involving a non-angiogenic function of VEGF signalling.