J Korean Assoc Oral Maxillofac Surg.  2014 Feb;40(1):3-10. 10.5125/jkaoms.2014.40.1.3.

Controlled release of nerve growth factor from heparin-conjugated fibrin gel within the nerve growth factor-delivering implant

• Lee JY
• Kim SM
• Kim MJ
• Lee JH

• KMID: 1960965
• DOI: http://doi.org/10.5125/jkaoms.2014.40.1.3

Abstract

OBJECTIVES
Although nerve growth factor (NGF) could promote the functional regeneration of an injured peripheral nerve, it is very difficult for NGF to sustain the therapeutic dose in the defect due to its short half-life. In this study, we loaded the NGF-bound heparin-conjugated fibrin (HCF) gel in the NGF-delivering implants and analyzed the time-dependent release of NGF and its bioactivity to evaluate the clinical effectiveness.
MATERIALS AND METHODS
NGF solution was made of 1.0 mg of NGF and 1.0 mL of phosphate buffered saline (PBS). Experimental group A consisted of three implants, in which 0.25 microL of NGF solution, 0.75 microL of HCF, 1.0 microL of fibrinogen and 2.0 microL of thrombin was injected via apex hole with micropipette and gelated, were put into the centrifuge tube. Three implants of experimental group B were prepared with the mixture of 0.5 microL of NGF solution, 0.5 microL HCF, 1.0 microL of fibrinogen and 2.0 microL of thrombin. These six centrifuge tubes were filled with 1.0 mL of PBS and stirred in the water-filled beaker at 50 rpm. At 1, 3, 5, 7, 10, and 14 days, 1.0 mL of solution in each tubes was collected and preserved at -20degrees C with adding same amount of fresh PBS. Enzyme-linked immunosorbent assay (ELISA) was done to determine in vitro release profile of NGF and its bioactivity was evaluated with neural differentiation of pheochromocytoma (PC12) cells.
RESULTS
The average concentration of released NGF in the group A and B increased for the first 5 days and then gradually decreased. Almost all of NGF was released during 10 days. Released NGF from two groups could promote neural differentiation and neurite outgrowth of PC12 cells and these bioactivity was maintained over 14 days.
CONCLUSION
Controlled release system using NGF-HCF gel via NGF-delivering implant could be an another vehicle of delivering NGF to promote the nerve regeneration of dental implant related nerve damage.

Keyword

Nerve growth factor-delivering implant; Nerve growth factor; Controlled release; Nerve regeneration

MeSH Terms

Animals
Dental Implants
Enzyme-Linked Immunosorbent Assay
Fibrin*
Fibrinogen
Half-Life
Nerve Growth Factor*
Nerve Regeneration
Neurites
PC12 Cells
Peripheral Nerves
Pheochromocytoma
Regeneration
Thrombin
Dental Implants
Fibrin
Fibrinogen
Nerve Growth Factor
Thrombin

Figure

• 

  Fig. 1 Design of nerve growth factor (NGF) delivering implant. A. Schematic design. B. Photographic images of NGF delivering implant.

• 

  Fig. 2 In vitro profile of NGF release from NGF-HCF gel loaded in NGF delivering implant at each time point of sampling (pg/mL). The amount of released NGF of two groups were increased and reached peak at days 5 and decreased. At day 3, released NGF of group A (459.666±302.739 pg/mL) was higher than that of group B (66.856±42.352 pg/mL), but there was no significance (Mann-Whitney U-test). (NGF: nerve growth factor, HCF: heparin-conjugated fibrin)

• 

  Fig. 3 Cumulative profile of released NGF from NGF-HCF gel loaded in NGF delivering implant (%). The cumulative release profile of NGF showed a similar pattern between two experimental groups over 14 days. For 10 days, almost all of NGF from NGF-HCF gel loaded in the NGF delivering implant was released. (NGF: nerve growth factor, HCF: heparin-conjugated fibrin)

• 

  Fig. 4 Microscopic images of the differentiated PC12 cells (×100). A. PC12 cells cultured with medium containing group A sample harvested at day 5. B. PC12 cells cultured with medium containing group B sample harvested at day 5. C. PC12 cells cultured with medium without NGF for 3 days (control group 1). D. PC12 cells cultured with medium without nerve growth factor for 6 days (control group 2). Scale bars=50 µm.

• 

  Fig. 5 Comparison of the ratio of differentiated PC12 cells in group A. A. Comparison with control group 1. The differentiation ratio at day 3 and 5 were significantly higher than that of control group 1. B. Comparison with control group 2. The ratio days 1, 3, 5, and 7 were higher than that of control group 2 with significance (*P<0.05, ***P<0.001; one-way ANOVA with Bonferroni's post hoc test). (NGF: nerve growth factor)

• 

  Fig. 6 Comparison of the ratio of differentiated PC12 cells in group B. A. Comparison with control group 1. B. Comparison with control group 2. The differentiation ratio of PC12 cells at days 3, 5, and 7 were significantly higher than those of control group 1 and 2 (*P<0.05, **P<0.01, ***P<0.001; one-way ANOVA with Bonferroni's post hoc test). (NGF: nerve growth factor)

• 

  Fig. 7 Comparison of the neurite length of differentiated PC12 cells in group A. A. Comparison with control group 1. B. Comparison with control group 2. No significant differences were found between group A, control group 1 and 2 (one-way ANOVA with Bonferroni's post hoc test). (NGF: nerve growth factor)

• 

  Fig. 8 Comparison of the neurite length of differentiated PC12 cells in group B. A. Comparison with control group 1. B. Comparison with control group 2. The average length of neurite of day 1 was significantly higher than those of control group 1 and 2 (*P<0.05; one-way ANOVA with Bonferroni's post hoc test). (NGF: nerve growth factor)

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