Histological and Immunohistochemical Evaluation of Rh-BMP2: Effect on Gingival Healing Acceleration and Proliferation of Human Epithelial Cells.

This study aims to histologically and immunohistochemically evaluate the effect recombinant human bone morphogenetic protein (rh-BMP2) injected in gingival tissue has on the acceleration of the epithelial migration from the wound edges and epithelial cell proliferation after implant surgery. MATERIAL AND METHODS: The study includes 20 patients who underwent bilateral implant surgeries in the premolar-molar region of the mandible, followed by guided bone regeneration. Each patient received an implant in both locations, but rh-BMP2 was only on the right side. At 9 days from the surgery, a gingival biopsy was performed 3 mm distally to the last implant. In total, 20 samples were collected from the left side (control group #1) and 20 from right (test group #1). This was repeated at a 4-month interval during healing abutment placements. Tissues were processed and stained with hematoxylin-eosin and then immunohistochemically for the expression of Ki-67 and further histological examination. RESULT: Complete closure of the epithelium with new cell formation was observed in the 55% test group and 20% control group after 9 days. At 4 months, although 100% samples of all groups had complete epithelial closure, the test group showed that the epithelial cells were more organized and mature due to the increased number of blood vessels. The average number of new epithelial cells was 17.15 ± 7.545 and 16.12 ± 7.683 cells per mm in test group, respectively, at 9 days and 4 months and 10.99 ± 5.660 and 10.95 ± 5.768 in control groups. CONCLUSION: Evident from histological observations, rh-BMP-2 can accelerate the closure of gingival wounds, the healing process of epithelial gingival tissue, and the formation of epithelial cells in patients undergoing dental implant treatment.

Effect of rh-BMP-2 in the Initiation of Neovascularization in Human Gingival Tissue: A Split-Mouth Clinical Study.

The aim of this study is to evaluate the effect on the initiation of new blood vessel formation of rh-BMP-2 administration in the human gingival tissue during bone regeneration surgery. MATERIAL AND METHODS: The randomized controlled clinical trial included twenty patients with bilateral partial edentulous of the mandibular premolar and molar region. Each patient received one implants on each side. Only one side received a 0.25 µg injection of rhBMP-2 into the gingival flap and grafted material during guided bone regeneration (GBR) for dental implantation. And the other side received GBR without injection. Three samples were collected from each patient as follows: one from the anterior area of the mandible (control group #1) collected at the time of all implant surgeries, and the two other samples during the placement of healing abutments at 4 months of follow-up, from treated side with rh-BMP-2 (test group) and untreated ones (control group #2). A total of 60 gingival samples were collected. Samples were stained with hematoxylin-eosin, and immunohistochemistry was performed with a vascular endothelial growth factor marker. The number of new vessels in each sample was counted. RESULT: Statistical analyses showed a significantly higher number of new vessels in the gingival tissue of the test group. CONCLUSIONS: Rh-BMP-2 injections into the gingival flap significantly improved new blood vessel formation.

Application of Escherichia coli-Derived Recombinant Human Bone Morphogenic Protein-2 to Unstable Spinal Fractures.

(1) Background: Recently, Escherichia coli-derived recombinant human bone morphogenetic protein-2 (E. coli-derived rhBMP-2) has been increasingly applied to different types of spinal surgeries and reported to achieve successful fusion. This pilot study aimed to evaluate the clinical efficacy and safety of rhBMP-2 in patients undergoing posterior instrumented fusions for unstable spinal fractures. (2) Methods: This study included ten consecutive patients undergoing spinal surgery using E. coli-derived rhBMP-2 with more than one year of follow-up. Radiologic outcomes were compared, including the average fracture healing period, local kyphosis correction, and clinical outcomes between preoperative and the last follow-up. (3) Results: The average time of radiographic union was 99.9 ± 45.4 (62-192) days, with an average use of 5.2 ± 3.9 months of anabolic agents. Radiologic parameters such as anterior vertebral height and vertebral wedge angle were significantly corrected postoperatively and at the last follow-up. Clinical outcomes other than leg pain were significantly improved after the surgery. In addition, four patients with preoperative neurologic deficits showed improved neurologic status. (4) Conclusions: Combined with the anabolic agents, applying E. coli-derived rhBMP-2 to the fractured vertebral body could be an effective surgical treatment for unstable spinal fractures. Further trials are needed to validate this result.

Enhanced Bone Regeneration in Variable-Type Biphasic Ceramic Phosphate Scaffolds Using rhBMP-2.

Our aim was to investigate the bone regeneration capacity of powder-type biphasic ceramic scaffold (BCP powder), block-type BCP (BCP block), and collagen-added block-type BCP (BCP collagen) with different concentrations of recombinant human bone morphogenetic protein 2 (rhBMP-2) in an animal model. Four rabbits were assigned to each of the following groups: no graft + rhBMP-2 (0.1/0.2 mg/mL), BCP powder + rhBMP-2 (0.1/0.2 mg/mL), BCP block + rhBMP-2 (0.1/0.2 mg/mL), and BCP collagen + rhBMP-2 (0.1/0.2 mg/mL), i.e., a total of 32 rabbits. Polycarbonate tubes (Φ 7 mm × 5 mm) for supporting scaffolds were fixed into a 7 mm round border. Subsequently, 0.1 mL of rhBMP-2 solutions with different concentrations was injected into the tubes. Both radiological and histomorphometric analyses showed that osteogenesis was not enhanced by increasing the concentration of rhBMP-2 in all groups at both 3 and 6 weeks. Radiological analysis showed that bone formation was higher in the BCP collagen group than in the BCP powder and BCP block groups at both rhBMP-2 concentrations at 3 weeks. rhBMP-2 enhanced bone formation; however, as the concentration increased, bone formation could not be enhanced infinitely. Collagen-added alloplastic graft material may be useful for mediating rapid bone formation in initial stages.

Ectopic osteogenesis effect of antigen-extracted xenogeneic cancellous bone graft with chitosan/rhBMP-2/bFGF sequential sustained-release nanocapsules.

To explore the ectopic osteogenesis effect of sequential sustained release application of recombinant human bone morphogenic protein-2 (rhBMP-2) and basic fibroblast growth factor (bFGF). Antigen-extracted xenogeneic cancellous bone coupled with growth factor-loaded chitosan nanocapsules were implanted in rats in intramuscular site in accordance with the following experimental pattern: group A: simultaneous burst release of rhBMP-2 and bFGF; group B: simultaneous sustained release of rhBMP-2 and bFGF; group C: preferential burst release of rhBMP-2, then sustained release of bFGF; group D: preferential burst release of bFGF, then sustained release of rhBMP-2; group E: sustained release of rhBMP-2 alone; group F: sustained release of bFGF alone, blank control group G: antigen-extracted xenogeneic cancellous bone graft only; negative control group H: not filled with anything. Specimens were obtained after executing the animals at 2 and 4 weeks for general observation and weighing, calcium content detection, micro-CT scanning and bone parameter measurement analysis, H&E staining, ALP staining and CD34 staining. The materials weight of A-2, B-2, C-2, A-4, B-4, C-4, D-4 and E-4 were significantly higher than that of preoperative materials ( P < 0.05). The concentration of calcium of group B-4 was the highest (414.7 ± 12.03 mg/dl). Micro-CT scanning and bone parameter measurement analysis showed that the values of bone mineral density and trabecular thickness of group A, B, D, E at 4 weeks were both higher than the ones at 2 weeks ( P < 0.05), and both the bone mineral density (367.52 ± 11.64 mg/cc) and the trabecular thickness (126.17 ± 11.36 µm) of group B-4 were the highest. H&E staining showed that a large region of calcified cartilage and haemopoietic tissues were newly formed, especially in group B-4. ALP staining and CD34 staining showed the most positive expression region in group B-4. Therefore, we conclude that simultaneous sustained release of rhBMP-2 and bFGF is the ideal way to release drug, and has better inducement of antigen-extracted xenogeneic cancellous bone graft.

Bone Morphogenic Protein-2 (rhBMP2)-Loaded Silk Fibroin Scaffolds to Enhance the Osteoinductivity in Bone Tissue Engineering.

There is an increasing demand for formulations of silk fibroin (SF) scaffolds in biomedical applications. SF was crosslinked via glutaraldehyde with osteoinductive recombinant human bone morphogenic protein-2 (rhBMP2) of different ratios viz. (i) 3% SF with no rhBMP2 (SF), (ii) 3% SF with equal amount of rhBMP2 (SF+BMP2), and (iii) 12% SF with 3% of rhBMP2 (4SF+BMP2), and these solutions were used in electrospinning-based fabrication of nanoscaffolds for evaluating increased osteoinductive potential of SF scaffolds with rhBMP2. Stress-strain relationship suggested there is no loss in mechanical strength of fibers with addition of rhBMP2, and mechanical strength of scaffold was improved with increase in concentration of SF. rhBMP2 association increased the water retention capacity of scaffold as evident from swelling studies. Viability of hMSCs was found to be higher in conjugated scaffolds, and scaffolds do not exhibit any cytotoxicity towards guest cells. Cells were found to have higher alkaline phosphatase activity in conjugated scaffolds under in vitro and in vivo conditions which establishes the increased osteoinductivity of the novel construct. The scaffolds were found to be effective for in vivo bone formation as well.

Surface modification of 3D-printed porous scaffolds via mussel-inspired polydopamine and effective immobilization of rhBMP-2 to promote osteogenic differentiation for bone tissue engineering.

UNLABELLED: For tissue engineering, a bio-porous scaffold which is applied to bone-tissue regeneration should provide the hydrophilicity for cell attachment as well as provide for the capability to bind a bioactive molecule such as a growth factor in order to improve cell differentiation. In this work, we prepared a three-dimensional (3D) printed polycaprolactone scaffold (PCLS) grafted with recombinant human bone morphogenic protein-2 (rhBMP2) attached via polydopamine (DOPA) chemistry. The DOPA coated PCL scaffold was characterized by contact angle, water uptake, and X-ray photoelectron spectroscopy (XPS) in order to certify that the surface was successfully coated with DOPA. In order to test the loading and release of rhBMP2, we examined the release rate for 28days. For the In vitro cell study, pre-osteoblast MC3T3-E1 cells were seeded onto PCL scaffolds (PCLSs), DOPA coated PCL scaffold (PCLSD), and scaffolds with varying concentrations of rhBMP2 grafted onto the PCLSD 100 and PCLSD 500 (100 and 500ng/ml loaded), respectively. These scaffolds were evaluated by cell proliferation, alkaline phosphatase activity, and real time polymerase chain reaction with immunochemistry in order to verify their osteogenic activity. Through these studies, we demonstrated that our fabricated scaffolds were well coated with DOPA as well as grafted with rhBMP2 at a quantity of 22.7±5ng when treatment with 100ng/ml rhBMP2 and 153.3±2.4ng when treated with 500ng/ml rhBMP2. This grafting enables rhBMP2 to be released in a sustained pattern. In the in vitro results, the cell proliferation and an osteoconductivity of PCLSD 500 groups was greater than any other group. All of these results suggest that our manufactured 3D printed porous scaffold would be a useful construct for application to the bone tissue engineering field. STATEMENT OF SIGNIFICANCE: Tissue-engineered scaffolds are not only extremely complex and cumbersome, but also use organic solvents which can negatively influence cellular function. Thus, a rapid, solvent-free method is necessary to improve scaffold generation. Recently, 3D printing such as a rapid prototyping technique has several benefits in that manufacturing is a simple process using computer aided design and scaffolds can be generated without using solvents. In this study, we designed a bio-active scaffold using a very simple and direct method to manufacture DOPA coated 3D PCL porous scaffold grafted with rhBMP2 as a means to create bone-tissue regenerative scaffolds. To our knowledge, our approach can allow for the generation of scaffolds which possessed good properties for use as bone-tissue scaffolds.

Gelatin Tight-Coated Poly(lactide-co-glycolide) Scaffold Incorporating rhBMP-2 for Bone Tissue Engineering.

Surface coating is the simplest surface modification. However, bioactive molecules can not spread well on the commonly used polylactone-type skeletons; thus, the surface coatings of biomolecules are typically unstable due to the weak interaction between the polymer and the bioactive molecules. In this study, a special type of poly(lactide-co-glycolide) (PLGA)-based scaffold with a loosened skeleton was fabricated by phase separation, which allowed gelatin molecules to more readily diffuse throughout the structure. In this application, gelatin modified both the internal substrate and external surface. After cross-linking with glutaraldehyde, the surface layer gelatin was tightly bound to the diffused gelatin, thereby preventing the surface layer gelatin coating from falling off within 14 days. After gelatin modification, PLGA scaffold demonstrated enhanced hydrophilicity and improved mechanical properties (i.e., increased compression strength and elastic modulus) in dry and wet states. Furthermore, a sustained release profile of recombinant human bone morphogenetic protein-2 (rhBMP-2) was achieved in the coated scaffold. The coated scaffold also supported the in vitro attachment, proliferation, and osteogenesis of rabbit bone mesenchymal stem cells (BMSCs), indicating the bioactivity of rhBMP-2. These results collectively demonstrate that the cross-linked-gelatin-coated porous PLGA scaffold incorporating bioactive molecules is a promising candidate for bone tissue regeneration.

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 (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.

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.