Evaluation of ethylene oxide, gamma radiation, dry heat and autoclave sterilization processes on extracellular matrix of biomaterial dental scaffolds

Abstract

Scaffolds used to receive stem cells are a promising perspective of tissue regeneration research, and one of the most effective solutions to rebuild organs. In the near future will be possible to reconstruct a natural tooth using stems cells, but to avoid an immune-defensive response, sterilize the scaffold is not only desired, but also essential to be successful. A study confirmed stem cells extracted from rat's natural teeth, and implanted into the alveolar bone, could differentiate themselves in dental cells, but the scaffold's chemistry, geometry, density, morphology, adherence, biocompatibility and mechanical properties remained an issue. This study intended to produce a completely sterilized dental scaffold with preserved extracellular matrix. Fifty-one samples were collected, kept in formaldehyde, submitted to partial demineralization and decellularization processes and sterilized using four different methods: dry heating; autoclave; ethylene-oxide and gamma-radiation. They were characterized through optical images, micro-hardness, XRD, EDS, XRF, SEM, histology and sterility test. The results evidenced the four sterilization methods were fully effective with preservation of ECM molecular arrangements, variation on chemical composition (proportion of Ca/P) was compatible with Ca/P proportional variation between enamel and dentine regions. Gamma irradiation and ethylene oxide presents excellent results, but their viability are compromised by the costs and technology's accessibility (requires very expensive equipment and/or consumables). Excepted gamma irradiation, all the sterilization methods more than sterilizing also reduced the remaining pulp. Autoclave presents easy equipment accessibility, lower cost consumables, higher reduction of remaining pulp and complete sterilization, reason why was considered the most promising technique.

Figure 1

SEM images, obtained at EVO MA ZEISS equipment from UNIFESP, did not evidence visual changes on dentine structure, but presented an apparent smoothing on enamel surface (GD1, GD2, GD3 and GD5), and also some cracks on the cement at GD2 and GD3 (the possibility of being caused by cutting process is not discarded).

Figure 2

Micro-hardness, measured at SHIMADZU HMV 2T, comparison between control group and sterilization methods, bars indicate the average hardness and the lines represent the standard deviation. Anova statistical analysis (Anova and graphics produced at excel 2013) indicates a significant association between the sterilization method and the micro-hardness, H value is highly significant 52.5432 (p = 0.00001).

Figure 3

(a) Measures at Bruker AXS equipment details the expected angles of the x-ray bean diffraction, CIFs obtained from ICSD—https://icsd.products.fiz-karlsruhe.de/), treated at Topas 4.2 also from Bruker, and plotted using Origin 2015. As it is a complex multi-atomic molecular structure, several angles are represented, but with higher incidence on angles near 26, 32–33, and 40 degrees, the reminiscence of angles positions means no molecular arrangement modification was detected. (b) XRF’s data obtained at Bruker’s S2 Ranger equipment the graphic was generated at Excel 2013—the sterilization methods present different effects on the composition balance (Ca/P), indicating GD3 (EO) affects more the Ca portion of Hydroxyapatite and GD2 (DH) interacts more intensively with P portion.

Figure 4

Histology analysis (inverted optical microscopy at AXIOVERT 40C using Axio Vision software)—structural modifications were not identified, although presented a variation on population, in special on GD1, GD2 and GD3 of colorants HE and MG; letter “d” represents dentine, and “e” enamel region. The software used to plot the images was Origin 2015.