• Microbiology and Biotechnology Letters
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• v.38 no.4
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• pp.420-427
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• 2010

A process for manufacturing virally-safe bovine amniotic membrane(BAM) has been developed for biological dressing. BAM was harvested from a healthy bovine placenta, and then the epithelium was removed. The remaining stromal layer was consecutively disinfected with 70% ethanol and 0.05% sodium hypochlorite. The stromal layer was incubated in a decellularization solution containing 0.25%(w/v) trypsin to remove the cellular components. The resulting acelluar BAM was lyophilized to preserve its biochemical and structural integrity. The BAM was packed and exposed to 25 kGy of gamma irradiation for sterilization purpose. Histological, electron microscopical, and biochemical observations showed that the acellualr BAM had intact structural integrity of three dimensional collagen fibers and contained several growth factors, accelerating wound healing, such as EGF (Epidermal growth factor), KGF (Keratinocyte growth factor), and FGF (Fibroblast growth factor). Bovine herpes virus (BHV), bovine viral diarrhoea virus (BVDV), bovine parainfluenza virus type 3 (BPIV-3), and bovine parvovirus (BPV) were chosen as the biological indicators for validation of viral safety of the acellular BAM. Samples from relevant stages of the production process were spiked with each virus and subjected to viral inactivation processes. Viruses were recovered from the samples and then titrated immediately. All the viruses tested were completely inactivated to undetectable levels within 1 h of 70% ethanol treatment. Enveloped viruses such as BHV, BVDV, and BPIV-3 were more effectively inactivated than BPV by 0.05% sodium hypochlorite treatment. BHV, BVDV, and BPIV-3 were completely inactivated to undetectable levels by 25 kGy of gamma irradiation. Also BPV was effectively inactivated by 25 kGy of gamma irradiation. The cumulative log reduction factors of BHV, BVDV, BPIV-3, and BPV were ${\geq}$13.30, ${\geq}$14.32, ${\geq}$15.22, and ${\geq}$7.57, respectively. These results indicate that the production process for acelluar BAM has a sufficient virus-reducing capacity to achieve a high margin of the virus safety.