CARBONATE APATITE-GELATIN MEMBRANE TO MODULATE AXONAL MICROENVIRONMENT AND REGENERATE PERIPHERAL NERVE

RETNO ARDHANI, drg. Ika Dewi Ana, M.Kes., Ph.D.; Prof. Yasuhiko Tabata, Ph.D. Tech, Ph.D. Med., Ph.D. Pharm; Prof. Kunio Ishikawa, Ph.D.

2017 | Disertasi | S3 Ilmu Kedokteran Gigi

Surgical intervention to bridge more than 5mm gap nerve defect is mandatory to promote regeneration. As nerve graft, autograft is considered as limited gold standard and allograft as well as xenograft as disadvantageous alternatives for immunosuppressant and cross-infection reasons. Thus, fabricated peripheral nervous devices are developed and biodegradable material is preferred because it was not promote post-regeneration injury on suturing line. In this study, peripheral nerve devices was prepared from biodegradable gelatin with carbonate apatite (CHA) as reinforcement to enhance mechanical strength. Incorporation of CHA in gelatin system was predicted to modulate calcium concentration in the environment. Based on the previous research, calcium concentration dynamics influenced axonal regeneration. Gelatin (CHA-00) and carbonated apatite-gelatin (CHA-05) membrane were prepared and characterized by evaluating Fourier Transform Infrared (FT-IR) spectra, scanning electron microscopy (SEM) graph, glucose permeation, air-water contact angle, swelling index, degradation rate, mechanical strength and calcium concentration in PBS and culture medium. Regeneration potential of CHA-00 and CHA-05 were studied from neurogenic differentiation of PC12 cell by evaluating acetylcholine (ACh) and dopamine release as well as neurite elongation. For the in vivo study, 18 Wistar rats were divided into control, CHA-00 and CHA-05 groups. Neuroetmesis injury with 5mm gap was performed on right sciatic nerve of rats in CHA-00 and CHA-05 groups. Weekly evaluation from 0 to 8 weeks after implantations were performed on autotomy behavior, sensory function by measuring withdrawal latency period and motor function by calculating sciatic functional index (SFI). After termination at week 8, histological study was performed to determine myelinated axon number and myelin thickness. Stereological method was applied to count myelinated axon number. From the FT-IR spectra, CO32- and PO43- groups were detected in CHA-05 peripheral nerve devices. Microstructure of CHA-00 and CHA-05 was similar on SEM evaluation. The addition of CHA on gelatin system was significantly increase diffusion percentage (p=0.046), decrease tensile strength (p=0.001) and increase compressive strength (p=0.05) but not significantly altered affinity percentage (p=0.121) and air-water contact angle (p=0.121). Swelling index and degradation rate of CHA-05 were lower than CHA-00. Initial higher calcium concentration in PBS and culture medium of PC12 cells were showed by CHA-05. The initial increase of calcium concentration in culture medium was not disrupting ACh release but increasing differentiation percentage and significantly promote longer neurite growth (p=0.020) of PC12 cells after 6 days culture. Meanwhile, dopamine release of PC12 cell in CHA-05 group was significantly lower than that in control (p=0.008) and CHA-00 group (p=0.004). Result of in vivo study showed lower autotomy score and incidence as well as no evidence of neuroma formation and allodynia in CHA-05 group. After 8 weeks implantation, withdrawal latency period and SFI were not significantly different between groups. Compared to baseline, SFI of CHA-05 group was not significantly different (p=0.065) after 8 weeks, but SFI of CHA-00 was significantly different (p=0.034). Considering the comparison of SFI after 8 weeks with baseline, CHA-05 promoted better motor function than CHA-00. Incorporation of CHA was not significantly increase myelinated axon number and myelin thickness (p=0.251 and p=0.964, respectively). It was concluded that incorporation of CHA increased mechanical strength of gelatin peripheral nerve device and induced initial higher calcium concentration that promoted better neurogenic differentiation of PC12 cells. However, in vivo regeneration potential of CHA-00 and CHA-05 were not significantly different.

Surgical intervention to bridge more than 5mm gap nerve defect is mandatory to promote regeneration. As nerve graft, autograft is considered as limited gold standard and allograft as well as xenograft as disadvantageous alternatives for immunosuppressant and cross-infection reasons. Thus, fabricated peripheral nervous devices are developed and biodegradable material is preferred because it was not promote post-regeneration injury on suturing line. In this study, peripheral nerve devices was prepared from biodegradable gelatin with carbonate apatite (CHA) as reinforcement to enhance mechanical strength. Incorporation of CHA in gelatin system was predicted to modulate calcium concentration in the environment. Based on the previous research, calcium concentration dynamics influenced axonal regeneration. Gelatin (CHA-00) and carbonated apatite-gelatin (CHA-05) membrane were prepared and characterized by evaluating Fourier Transform Infrared (FT-IR) spectra, scanning electron microscopy (SEM) graph, glucose permeation, air-water contact angle, swelling index, degradation rate, mechanical strength and calcium concentration in PBS and culture medium. Regeneration potential of CHA-00 and CHA-05 were studied from neurogenic differentiation of PC12 cell by evaluating acetylcholine (ACh) and dopamine release as well as neurite elongation. For the in vivo study, 18 Wistar rats were divided into control, CHA-00 and CHA-05 groups. Neuroetmesis injury with 5mm gap was performed on right sciatic nerve of rats in CHA-00 and CHA-05 groups. Weekly evaluation from 0 to 8 weeks after implantations were performed on autotomy behavior, sensory function by measuring withdrawal latency period and motor function by calculating sciatic functional index (SFI). After termination at week 8, histological study was performed to determine myelinated axon number and myelin thickness. Stereological method was applied to count myelinated axon number. From the FT-IR spectra, CO32- and PO43- groups were detected in CHA-05 peripheral nerve devices. Microstructure of CHA-00 and CHA-05 was similar on SEM evaluation. The addition of CHA on gelatin system was significantly increase diffusion percentage (p=0.046), decrease tensile strength (p=0.001) and increase compressive strength (p=0.05) but not significantly altered affinity percentage (p=0.121) and air-water contact angle (p=0.121). Swelling index and degradation rate of CHA-05 were lower than CHA-00. Initial higher calcium concentration in PBS and culture medium of PC12 cells were showed by CHA-05. The initial increase of calcium concentration in culture medium was not disrupting ACh release but increasing differentiation percentage and significantly promote longer neurite growth (p=0.020) of PC12 cells after 6 days culture. Meanwhile, dopamine release of PC12 cell in CHA-05 group was significantly lower than that in control (p=0.008) and CHA-00 group (p=0.004). Result of in vivo study showed lower autotomy score and incidence as well as no evidence of neuroma formation and allodynia in CHA-05 group. After 8 weeks implantation, withdrawal latency period and SFI were not significantly different between groups. Compared to baseline, SFI of CHA-05 group was not significantly different (p=0.065) after 8 weeks, but SFI of CHA-00 was significantly different (p=0.034). Considering the comparison of SFI after 8 weeks with baseline, CHA-05 promoted better motor function than CHA-00. Incorporation of CHA was not significantly increase myelinated axon number and myelin thickness (p=0.251 and p=0.964, respectively). It was concluded that incorporation of CHA increased mechanical strength of gelatin peripheral nerve device and induced initial higher calcium concentration that promoted better neurogenic differentiation of PC12 cells. However, in vivo regeneration potential of CHA-00 and CHA-05 were not significantly different.

Kata Kunci : carbonated apatite, gelatin, calcium, PC12, sciatic nerve, 5mm gap