Effect of norbixin-based poly(hydroxybutyrate) membranes on the tendon repair process after tenotomy in rats

Abstract Purpose: To determine the efficacy of norbixin-based poly(hydroxybutyrate) (PHB) membranes for Achilles tendon repair. Methods: Thirty rats were submitted to total tenotomy surgery of the right Achilles tendon and divided into two groups (control and membrane; n = 15 each), which were further subdivided into three subgroups (days 7, 14, and 21; n = 5 each). Samples were analyzed histologically. Results: Histological analysis showed a significant reduction in inflammatory infiltrates on days 7, 14 (p < 0.0001 for both), and 21 (p = 0.0004) in the membrane group compared to that in the control group. There was also a significant decrease in the number of fibroblasts in the control group on days 7, 14 (p < 0.0001), and 21 (p = 0.0032). Further, an increase in type I collagen deposition was observed in the membrane group compared to that in the control group on days 7 (p = 0.0133) and 14 (p = 0.0107). Conclusion: Treatment with norbixin-based PHB membranes reduces the inflammatory response, increases fibroblast proliferation, and improves collagen production in the tendon repair region, especially between days 7 and 14.

Effects of bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) release from polylactide-poly (ethylene glycol)-polylactide (PELA) microcapsule-based scaffolds on bone

Multiple growth factors can be administered to mimic the natural process of bone healing in bone tissue engineering. We investigated the effects of sequential release of bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) from polylactide-poly (ethylene glycol)-polylactide (PELA) microcapsule-based scaffolds on bone regeneration. To improve the double emulsion/solvent evaporation technique, VEGF was encapsulated in PELA microcapsules, to which BMP-2 was attached. The scaffold (BMP-2/PELA/VEGF) was then fused to these microcapsules using the dichloromethane vapor method. The bioactivity of the released BMP-2 and VEGF was then quantified in rat mesenchymal stem cells (rMSCs). Immunoblotting analysis showed that BMP-2/PELA/VEG promoted the differentiation of rMSCs into osteoblasts via the MAPK and Wnt pathways. Osteoblast differentiation was assessed through alkaline phosphatase expression. When compared with simple BMP-2 plus VEGF group and pure PELA group, osteoblast differentiation in BMP-2/PELA/VEGF group significantly increased. An MTT assay indicated that BMP-2-loaded PELA scaffolds had no adverse effects on cell activity. BMP-2/PELA/VEG promoted the differentiation of rMSCs into osteoblast via the ERK1/2 and Wnt pathways. Our findings indicate that the sequential release of BMP-2 and VEGF from PELA microcapsule-based scaffolds is a promising approach for the treatment of bone defects.

In vitro analysis of a local polymeric device as an alternative for systemic antibiotics in Dentistry

Abstract The development of a biodegradable material with antimicrobial properties for local applications is required in the prevention and treatment of infectious diseases. The objective of this study was to produce blends of poly-L-lactide acid (PLLA) synthetic polymer associated with several antimicrobials, as an alternative in the prevention and treatment of infections, as well as to evaluate its cytotoxicity, release of antimicrobials and inhibit bacteria growth. Blends of PLLA added with 20% Amoxicillin, Metronidazole, Clindamycin or Azithromicyn were used to produce Films (F) or Meshs (M) by casting and electrospinning methods, respectively. Standardized discs of the films and meshs were stored in buffer solutions (pH 5 or 7.4) and aliquots were analyzed by high performance chromatography (HPLC) during 168 hours. Cytotoxicity on human gingival fibroblasts was tested after 24, 48 and 72h by MTT reaction. The antimicrobial capacity was determined against P. gingivalis and S. pyogenes. The specimens were weighed after 3 and 6 months of storage for degradation analysis. SEM was performed to control interfaces and degradation. Antimicrobials presented a continuous and exponential drug release. Analysis showed that both M and F were able to inhibit S. pyogenes and P. gingivalis growth, indicating the release of active antimicrobial agents. The products were not toxic to the fibroblasts. Amoxicillin-film showed more degradation than PLLA at both pHs (p < 0.05), whereas Azithromycin-meshes were more degraded than PLLA at pH 7.4 (p < 0.05). PLLA association with antimicrobials is biocompatible and may represent a potential tool for the local delivery of antimicrobials.

Effects of Polycaprolactone-Tricalcium Phosphate, Recombinant Human Bone Morphogenetic Protein-2 and Dog Mesenchymal Stem Cells on Bone Formation: Pilot Study in Dogs

PURPOSE: The aim of this study was to evaluate the survival, proliferation, and bone formation of dog mesenchymal stem cells (dMSCs) in the graft material by using Polycaprolactone-tricalcium phosphate (PCL-TCP), auto-fibrin glue (AFG), recombinant human bone morphogenetic protein-2 (rhBMP-2), and dMSCs after a transplantation to the scapula of adult beagle dogs. MATERIALS AND METHODS: The subjects were two beagle dogs. Total dose of rhBMP-2 on each block was 10 microg with 50 microg/mg concentration. The cortical bone of the scapula of the dog was removed which was the same size of PCL-TCP block (Osteopore International Pte, Singapore; 5.0x5.0x8.0 mm in size), and the following graft material then was fixed with orthodontic mini-implant, Dual-top(R) (Titanium alloy, Jeil Co. Seoul, Korea). Four experimental groups were prepared for this study, Group 1: PCL-TCP + aFG; Group 2: PCL-TCP + aFG + dMSCs; Group 3: PCL-TCP + aFG + dMSCs + rhBMP-2; Group 4: PCL-TCP + aFG + dMSCs + rhBMP-2 + PCL membrane. The survival or proliferation of dMSCs cells was identified with an extracted tissue through a fluorescence microscope, H-E staining and Von-Kossa staining in two weeks and four weeks after the transplantation. RESULTS: The survival and proliferation of dMSCs were identified through a fluorescence microscope from both Group 1 and Group 2 in two weeks and four weeks after the transplantation. Histological observation also found that the injected cells were proliferating well in the G2, G3, and G4 scaffolds. CONCLUSION: This study concluded that bone ingrowth occurred in PCL-TCP scaffold which was transplanted with rhBMP-2, and MSCs did not affect bone growth. More sufficient healing time would be needed to recognize effects of dMSCs on bone formation.

Guided bone regeneration with beta-tricalcium phosphate and poly Llactide-co-glycolide-co-epsilon-caprolactone membrane in partial defects of canine humerus

This study was performed to evaluate the effect of betatricalcium phosphate and poly L-lactide-co-glycolide-coepsilon- caprolactone (TCP/PLGC) membrane in the repair of partial bone defects in canine proximal humerus. Three adult mixed-breed dogs were used during the experimental period. The length of the defect was quarter of the full length of humerus, and width of the defect was quarter of middle diameter of the lateral aspect of humerus. The humeri of each dog were divided into treatment (TCP/ PLGC) and control groups. The defect was covered with TCP/PLGC membrane in treatment group. To evaluate regeneration of the bone, computerized tomography (CT) and histopathologic examination were performed. The radiopaque lines were appeared at the original defect sites in TCP/PLGC group but below the original site in control at 4th week. Radiopacity and thickness of the defect sites, and radiopaque lines were more increased at 8th week than those of 4th week. Histopathologic findings revealed fibrous connective tissue migration into the defect and the migration inhibited the structure of new cortex to be placed in the original level in control whereas new cortex growth was found in the level of original line in TCP/ PLGC group. However, the new cortical bone in the TCP/ PLGC group was thinner and less organized than the adjacent intact cortex, and the amount of new cancellous bones were also scanty. The result suggested that TCP/ PLGC membrane is a good guided bone regeneration material to restore the original morphology of humerus in partial defect.

Immunization by subcutaneous implants of polyester-polyurethane sponges coupled with antigen

A new protocol is described for immunization of outbred Swiss mice. The procedure is based on subcutaneous implantation of antigen-coupled polyester-polyurethane sponges cut into disks of 10 mm in diameter vs 2 mm in thickness. Antigen coupling was performed by overnight incubation of the sponge with a solution of ovalbumin (Ova) (2 mg/ml) diluted in sodium carbonate buffer, pH 9.6. The amount of ovalbumin that was taken up by the sponge was between 71.4 to 82.5 µg. This was estimated by comparing the Ova absorbance at 280 nm in coating buffer solutions before and after incubation. To compare the efficiency of the proposed method, experimental groups immunized with the antigen in the presence of adjuvants (10 µg in Al(OH)3 or 100 µg in complete Freund's adjuvant (CFA)) were run in parallel. The data obtained after the 3rd week of immunization indicate that both cellular and humoral immune responses were achieved. These were assayed by antigen-induced footpad swelling and ELISA (specific antibodies), respectively. The levels of both immune responses elicited were similar to the responses observed in mice immunized with ovalbumin in the presence of Al(OH)3. The method might represent an advantage when immunizing with pathogenic antigens. Preliminary experiments have suggested that the antigen remains immobilized or bound to the sponge for a long period of time, since there is an increment on the cell population inside the sponges after boosting the animals. If so, the undesirable effects of immunization would be reduced.