Evaluation of Osteogenic Potentials of Avian Demineralized Bone Matrix in the Healing of Osseous Defects in Pigeons.

OBJECTIVES: To evaluate avian allogeneic demineralized bone matrix (DBM) in the healing of long bone defects as a function of geometry and time in a pigeon model. STUDY DESIGN: Experimental. ANIMALS: Adult rock pigeons (n = 60). METHODS: Midshaft ulnar osseous defects were grafted with 2 geometric forms of DBM (tubular vs. chipped) and stabilized with a hybrid fixator. Autologous chips of sternal keel were used in a third group as control. Outcomes were evaluated by radiography and histology/histomorphometry at 4, 8, 12, and 24 weeks postoperatively. RESULTS: Despite an early rapid healing response, autografts plateaued (histologic score and new bone area) by 8 weeks with no significant improvement afterwards. Conversely, allogeneic DBM implants demonstrated continuous temporal improvement in bone healing, and tubular DBM finally outpaced autograft implants after week 12 with values for metrics achieving statistical significance by week 24. Chip DBM was inferior to tubular DBM and autograft. CONCLUSIONS: Avian DBM is osteogenic, biocompatible, and safe in orthotopic sites with potential usefulness in avian bone grafting. Implant geometry (shape and size) affects such osteogenic potentials.

Caprine pancreatic islet xenotransplantation into diabetic immunosuppressed BALB/c mice.

BACKGROUND: Type 1 diabetes mellitus is a devastating disease for which there is currently no cure, but only lifetime management. Islet xenotransplantation is a promising technique for the restoration of blood glucose control in patients with diabetes mellitus. The purpose of this study was to explore the potential use of caprine (goat) islet cells as xenogeneic grafts in the treatment for diabetes in a mouse model. METHODS: Caprine pancreases were harvested and transported to the laboratory under conditions optimized to prevent ischemia. Islets were isolated, purified, and tested for functionality. Caprine islets (2000 islet equivalent) were transplanted beneath the kidney capsules of diabetic BALB/c mice under thalidomide-induced immunosuppression. Blood glucose and insulin levels of grafted mice were evaluated by glucometer and enzyme-linked immunosorbent assay kit, respectively. The functionality and quality of caprine pancreatic islet grafts were assessed by intraperitoneal glucose tolerance tests. RESULTS: The viability of purified islet cells exceeded 90%. Recipient mice exhibited normoglycemia (<11 mM glucose) for 30 days. In addition, weight gain negatively correlated with blood glucose level. The findings verified diabetes reversal in caprine islet recipient mice. A significant drop in non-fasting blood glucose level (from 23.3 ± 5.4 to 8.04 ± 0.44 mM) and simultaneous increase in serum insulin level (from 0.01 ± 0.001 to 0.56 ± 0.17 µg/l) and body weights (from 23.64 ± 0.31 to 25.85 ± 0.34 g) were observed (P < 0.05). Immunohistochemical analysis verified insulin production in the transplanted islets. CONCLUSIONS: Purified caprine islets were demonstrated to successfully sustain viability and functionality for controlling blood glucose levels in an immunosuppressed mouse model of diabetes. These results suggest the use of caprine islets as an addition to the supply of xenogeneic islets for diabetes research.

Qualitative and quantitative evaluation of avian demineralized bone matrix in heterotopic beds.

OBJECTIVE: To evaluate the osteogenic potential of avian demineralized bone matrix (DBM) in the context of implant geometry. STUDY DESIGN: Experimental. ANIMALS: Rock pigeons (n = 24). METHODS: Tubular and chipped forms of DBM were prepared by acid demineralization of long bones from healthy allogeneic donors and implanted bilaterally into the pectoral region of 24 pigeons. After euthanasia at 1, 4, 6, 8, 10, and 12 weeks, explants were evaluated histologically and compared by means of quantitative (bone area) and semi quantitative measures (scores). RESULTS: All explants had new bone at retrieval with the exception of tubular implants at the end of week 1. The most reactive part in both implants was the interior region between the periosteal and endosteal surfaces followed by the area at the implant-muscle interface. Quantitative measurements demonstrated a significantly (P = .012) greater percentage of new bone formation induced by tubular implants (80.28 ± 8.94) compared with chip implants (57.64 ± 3.12). There was minimal inflammation. CONCLUSIONS: Avian DBM initiates heterotopic bone formation in allogeneic recipients with low grades of immunogenicity. Implant geometry affects this phenomenon as osteoconduction appeared to augment the magnitude of the effects in larger tubular implants.

Insulin secreted from genetically engineered intestinal cells reduces blood glucose levels in diabetic mice.

Poorly controlled diabetes mellitus can result in serious complications. Gene therapy is increasingly being considered as an alternative approach to treat diabetes, because of its ability to induce physiological insulin secretion and it allows patients to escape insulin injections. The properties of gut K and L-cells, including glucose sensitivity, the ability to process insulin and a regulated secretion pathway support their use as surrogate ß-cells. Previous in vitro studies have provided sufficient evidence supporting the use of these cells for gene therapy studies. Therefore, we examined the ability of K and L-cells to produce insulin in diabetic mice. Chitosan nanoparticles were used to transfer the insulin gene into intestinal cells via oral administration. The efficiency of chitosan as a gene vehicle was investigated through the use of reporter gene. Insulin mRNA and protein expression levels were measured by RT-PCR and ELISA, respectively. Blood glucose testing revealed that this treatment reduced glucose levels in diabetic mice. The decrease in blood glucose level in the first week of treatment was greater in mice with K-cell specific insulin expression compared with mice with L-cell-specific insulin expression. These results indicate that inducing insulin secretion in K-cells conferred a quicker response to gene therapy.

Engineering an L-cell line that expresses insulin under the control of the glucagon-like peptide-1 promoter for diabetes treatment.

BACKGROUND: Diabetes mellitus is a complicated disease with a pathophysiology that includes hyperinsulinemia, hyperglycemia and other metabolic impairments leading to many clinical complications. It is necessary to develop appropriate treatments to manage the disease and reduce possible acute and chronic side effects. The advent of gene therapy has generated excitement in the medical world for the possible application of gene therapy in the treatment of diabetes. The glucagon-like peptide-1 (GLP-1) promoter, which is recognised by gut L-cells, is an appealing candidate for gene therapy purposes. The specific properties of L-cells suggest that L-cells and the GLP-1 promoter would be useful for diabetes therapy approaches. RESULTS: In this study, L-cells were isolated from a primary intestinal cell line to create suitable target cells for insulin expression studies. The isolated cells displayed L-cell properties and were therefore used as an L-cell surrogate. Next, the isolated L-cells were transfected with the recombinant plasmid consisting of an insulin gene located downstream of the GLP-1 promoter. The secretion tests revealed that an increase in glucose concentration from 5 mM to 25 mM induced insulin gene expression in the L-cells by 2.7-fold. Furthermore, L-cells quickly responded to the glucose stimulation; the amount of insulin protein increased 2-fold in the first 30 minutes and then reached a plateau after 90 minutes. CONCLUSION: Our data showed that L-cells efficiently produced the mature insulin protein. In addition, the insulin protein secretion was positively regulated with glucose induction. In conclusion, GLP-1 promoter and L-cell could be potential candidates for diabetes gene therapy agents.

Heterotopic implantation of autologous bone marrow in rock pigeons (Columba livia): possible applications in avian bone grafting.

Autologous bone marrow, alone or as a composite marrow graft, has received much attention in various species. To assess the potential osteogenicity of autologous, extramedullary bone marrow implants in an avian model, 24 adult pigeons (Columba livia) were given intramuscular implantations of autologous marrow aspirated from the medial tibiotarsus. Birds were euthanatized at 1, 4, 6, 8, 10, and 12 weeks after surgery to evaluate whether ectopic bone had formed at the implant sites. Primary evaluations by in situ radiography and postmortem histologic examinations showed no evidence of bone formation. Further evaluation with histologic scores and histomorphometry revealed a significantly increased rate of angiogenesis at the implant sites by the sixth and tenth week postimplantation (P < .05). No significant differences between the treatment and control sites were present at any other endpoints. Results of this study show that, although autologous bone marrow lacks heterotopic osteogenic potentials in this avian model, it could still function as a useful adjunct to routine bone grafting techniques because of its unique capabilities to promote early angiogenesis.

Isolation, density purification, and in vitro culture maintenance of functional caprine islets of Langerhans as an alternative islet source for diabetes study.

BACKGROUND: Insufficient availability of human donors makes the search for alternative source of islet cells mandatory for future developments in pancreatic transplantation. The present study investigates the potential of caprine as an alternative source of pancreatic islets. The objectives of the study were to optimize techniques for caprine islet isolation and purification for culture establishment, and to subsequently assess their viable and functional potential. METHODS: Caprine pancreatic tissues were collected from a local slaughterhouse and prior transported to the laboratory by maintaining the cold chain. Islets were obtained by a collagenase-based digestion and optimized isolation technique. Islet cell purity and viability were determined by dithizone and trypan blue staining, respectively. Islet clusters of different sizes were positively identified by staining methods and demonstrated 90% viability in the culture system. Following static incubation, an in vitro insulin secretion assay was carried out and analyzed by ELISA. RESULTS: The islets remained satisfactorily viable for 5 days in the culture system following regular media changes. The current study has successfully optimized the isolation, purification and culture maintenance of caprine islets. CONCLUSION: The successful yield, viability and functionality of islets isolated from the optimized protocol provide promising potential as an alternative source of islets for diabetes and transplantation researches.