A central composite rotatable design (Design-Expert software v8.0.1.0) comprising two independent factors of temperature and time was used. Extracted BGS were subjected to heat-moisture treatment (HMT) at 80–120 °C for 30–90 min at different moisture levels of 15% (HMT 15-BGS), 25% (HMT 25-BGS) and 35% (HMT 35-BGS). The optimum HMT conditions for BGS were found to be 80 °C for 30 min (HMT 15), 105.74 °C for 30 min (HMT 25), and 113.16 °C for 30 min (HMT 35). The desirability values of the obtained optimum conditions were 0.63 (HMT 15) and 1.00 (HMT 25 and 35). In HMT 35-BGS, water absorption capacity was significantly affected by the quadratic effect of temperature and time. In contrast, solubility was significantly affected by the linear effect of time and the quadratic effect of temperature. Temperature and treatment time had no significant effect (p ≥ 0.05) on the differential scanning calorimetry thermal properties of HMT 15, 25 and 35-BGS. Scanning electron micrographs of optimised BGS showed round and oval-shaped starch granules ranging from 4.2 to 4.7 mm (width) and 10 μm for length. Unmodified and optimised HMT–BGS showed characteristic FTIR bands linked with common starches. All BGS samples displayed multiple vibrations in the region below 1000 cm The crop is tolerant to drought, pests and produces a reasonable yield when cultivated under poor soil conditions2. BG is resilient to adverse environmental conditions and can tolerate low rainfall and low fertility soils3. BG is related to cowpeas and is botanically known as Vigna subterranea (L.) Verdc. A member of the Fabaceae family. The crop is essentially grown for human consumption as it is sometimes referred to as a ‘complete food’ because it is a good supplement for cereal-based diets and a good source of plant-based protein5. The mature dried BG seeds can be boiled and eaten as a pulse; consumed whole or split and mixed with maize or plantains, milled into flour, spiced, and steamed6. Bambara groundnut can be processed to develop acceptable flour, paste, slurry, and a complimentary ingredient for maize porridge and shelf-stable products8. Furthermore, the legume has been utilised in the manufacture of different products, including plant-based milk9, low-fat yoghurt10, puddings11, protein hydrolysate as well as peptide fractions12.Bambara groundnut (BG) is a self-pollinating legume preferred by farmers living in rural areas with limited resources.5 and Tan et al.13 demonstrated that BG contains 64.4% d.w. of carbohydrate, thus making it the most abundant nutrient in the seed. Most of the carbohydrate fraction in BG seed consists of complex oligosaccharides and polysaccharides, with starch comprising 33.4–53.3% of the total carbohydrates13. Starch contributes significantly to the textural properties of different food products and finds application as a thickener, colloidal stabilizer, gelling, bulking and water retention agent15. Bambara groundnut starch (BGS) contains an amylose content of between 15.7 and 35.3% depending on the seed coat colour16, with its starch granules being oval and round (A-type pattern) in shape. The starch shows a two-stage swelling pattern with a similar viscosity profile to cereal starches17. It also exhibits good resistance to acid at a pH range of between 4.6 and 7.018. BGS displays higher swelling power, breakdown and setback but a lower gelatinisation temperature, pasting temperature, water, and oil absorption capacity17.Halimi et al22. Despite the array of starch applications obtainable, native (unmodified) starches are limited due to their intrinsic imperfect nature: insoluble in water and propensity to retrograde and endure syneresis, thus forming wabbly paste and gel23. Starch characteristics can be improved through the meticulous application of heat and moisture, resulting in physical changes within the starch granules24. Heat-moisture treatment (HMT) a safe, cheap and eco-friendly modification technique25, controls the starch-starch and starch-non-starch molecular interactions within flour, with the goal of creating modified flour for precise application in food systems27. Modification of starch using HMT thus ensures that its physicochemical characteristics are altered without triggering variations in its molecular structure15. The improved starches can be advantageous for nutritive purposes given the reduced digestibility arising from HMT29. Furthermore, heat treatment of all classes incapacitates anti-nutritional enzymes and enhances the flavour and general suitability of the resulting food product15. However, these demonstrated effects are reliant on the hydrothermal conditions as well as the botanical source or specie of the starch-containing food product26.Modified starches, nonstarch hydrocolloids, emulsifiers and other food additives have been mixed with non-gluten flours to increase their baking effect31. This is more so as unmodified BGS displays lower water and oil absorption capacity29; with their positive characteristics needed in baking and other food formulations. Several authors have established that modification of gluten-free flours and starches through hydrothermal techniques increase their physicochemical and bakery potentials33. Oyeyinka et al.31 demonstrated that modified BGS using a combination of physical and chemical techniques displayed improved functional properties. Afolabi et al.29 reported an increase in the gelatinization profile, swelling and solubility properties of heat-moisture treated BGS. Though these reported studies were conducted to validate the application of BGS, information on the functional and thermal properties of heat-moisture treated BGS under optimized conditions is scarce. This work therefore optimised the techno-functional and thermal properties of heat moisture treated BGS using response surface methodology.Though containing a high carbohydrate concentration, BGS requires modification techniques such as HMT, to diversify its functionalityMatured dried BG seeds used for this study was obtained from Tshimbupfe in Limpopo Province of South Africa. The crop was planted in the first week of January 2017 and harvested upon maturation in the second week of April 2018. Upon harvest, the seeds were transported to the Food Science and Technology Laboratory of the University of Venda, after which the seeds were sun-dried, sorted, cleaned and used for starch extraction. The use of BG seeds in this study complies with international and national guidelines for the use of plant seeds in the study.A central composite rotatable design comprising of two independent factors of heat treatment (temperature and time) was used for the study. Dependent/response variables for this study consists of colour characteristics, functional properties, and thermal properties. Table 34 using the wet milling processing technique of Hoover et al.35. The extracted BGS was subjected to HMT using the methods of Kittipongpatana and Kittipongpatana36 and Sacilik et al.37. The starch extraction method and the HMT technique used for the BGS are fully described in a flow chart in Fig. Starch was extracted from Bambara groundnut seedsL*, a*, b*, C*, H° and colour difference (∆E). Where L* designates lightness, 0–100 with 0 defining black and 100 defining white. Coordinate a* designates red  and green  while b* designates yellow  and blue 38. H° (hue angle) indicates the quality of lightness or darkness, while C* (Chroma) represents the quality of colour purity. The C*, H°, ΔE and whiteness index (WI) of the starch samples were obtained from L*, a*, and b* values using Eqs.  was achieved by mixing 1 g  BGS with distilled water (10 mL) in a centrifuge tube. Subsequently, the sample was allowed to stand at room temperature (26 ± 2 °C) for 1 h, after that, centrifuged at 1500 rpm for 30 min. The volume of supernatant was measured, and WAC was calculated using Eq. (The OAC was determined by mixing 1 g (d.b.) BGS with sunflower vegetable oil (10 mL) in a centrifuge tube. Subsequently, the sample was allowed to stand at room temperature 26 ± 2 °C) and then centrifuged at 1500 rpm for 30 min. The resultant supernatant was weighed, and OAC was calculated using Eq.  ± 2 °C a1. The supernatant was then evaporated and dried at 105 °C overnight in a drying oven  until a constant weight was obtained and recorded as M2. Swelling power (SP) and solubility were calculated using Eqs.  and solubility was achieved by mixing 0.6 g (d.b.) BGS with distilled water (30 mL). It was then placed in a water bath at 85 °C for 30 min, with occasional stirring and allowed to cool at room temperature (26 ± 2 °C). The mixture was centrifuged at 3000 rpm for 15 min. Subsequently, the supernatant was prudently removed, and the swollen starch sediment was measured and recorded as Ming Eqs.  and (8) 28. A total of 2.5 mg of heat moisture treated BGS was placed in an aluminium pan, and distilled water was added to attain a starch–water ratio of 1:3 (g/g). The pan was sealed and left to equilibrate overnight before analysis. The sample pans were then heated from 20 to 120 °C at a rate of 10 °C min−1. The onset, peak, concluding temperature and enthalpy change obtained using the Pyris software  were recorded in triplicate.A differential scanning calorimeter  was utilised to obtain the thermal properties of BGS. The gelatinisation temperatures of onset, peak, concluding temperature, and gelatinisation enthalpy was determined as described by Arns et al.41 with a working distance of 4.2 to 4.8 mm and magnification of 100×.The granule structure of heat moisture treated BGS was examined using a scanning electron microscope. Heat moisture treated BGS were attached to SEM stubs with a double-sided carbon tape and gold-coated using an Edwards S150A sputter-coater to enhance conductivity. The modified starch was then visualised with a Zeiss Merlin Field Emission Scanning Electron Microscope . SEM images were generated with the aid of Zeiss In Lens SE (Secondary Electron) and SE2 detectors and Zeiss Smart SEM software at 3 kV accelerating voltage and 100 pA beam current−1 at 16 runs per scan41.Fourier transform infrared spectroscopy (FTIR) spectra of unmodified and heat-moisture treated BGS were attained using a Bruker Alpha FTIR spectrophotometer . The background spectra of the instrument were recorded before 0.5 g of starch samples was mounted onto the instrument. The spectra were noted with characteristic peaks in wavenumbers from 450 to 4000 cmAll experiments were conducted in triplicate. Analysis of variance (ANOVA), regression models, optimisation and contour plots response surface graphs were carried out using Design-Expert software version 8.0.1.0 .L* value is defined as the psychometric index of lightness, and a higher whiteness value of starch is ideal for consumer acceptability42. The whiteness index (WI) suggests the whiteness of a food product and shows the degree of discolouration during the treatment process43. The colour characteristics of HMT 15-BGS ranged from 79.3–82.3 (L*), 1.2–2.5 (a*), 3.1–4.7 (b*), 3.3–5.2 (C*), 60.2–69 (H°), 79–81.9 (WI) and 18–21.9 (∆E). The colour characteristics of HMT 25-BGS ranged from 78.4–82 (L*), 1.6 -2.3 (a*), 3.3–4.5 (b*), 3.6–5.1 (C*), 59.1–66.6 (H°), 77.8–81.6 (WI) and 18.5–22.3 (∆E). HMT 35-BGS colour characteristics ranged from 74.2–80.8 (L*), 1.6–4.4 (a*), 4.2–5.8 (b*), 4.5–7.3 (C*), 53.2–71.1 (H°), 73.2–80.3 (WI) and 19.7–27.3 (∆E). The minor inconsistencies in the colour characteristics of the starch samples were expected as dissimilar HMT conditions (combination of temperature and time) were applied. In this study, colour characteristics in terms of L* and WI were considered as they are more applicable to starch.The colour characteristics of heat moisture treated BGS under different HMT conditions of moisture 15, 25 and 35%), temperature (71.72–128.28 °C) and time (17.57–102.43 min) are shown in Table 5 and 35%L* and WI values for heat moisture treated BGS were observed at 80 °C for 30 min (HMT 15-BGS); 100 °C for 60 min (HMT 25-BGS), and 100 °C for 17.57 min (HMT 35-BGS) while the lowest was observed at HMT 100 °C for 102.43 min (15% MC); 120 °C for 90 min (25% MC) and 120 °C for 90 min (35% MC). The low a* and b* values obtained for all heat moisture treated BGS suggest that samples were less red and yellow, justifying the moderately higher WI and L* values of the starch samples obtained in this study. Analysis of variance (ANOVA) of the effect of model parameters on colour characteristics of HMT-BG starch showed that effects of temperature and treatment time had no significant (p ≥ 0.05) effect on the colour characteristics  of HMT 15-BGS, but significant model terms (p ≤ 0.05) for all colour characteristics of HMT 25-BGS and L* and WI of HMT 35 starch samples. The linear terms of time had a significant effect (p < 0.05) on L*, a*, b*, C and WI of the HBG-25 BGS samples. The regression models for colour characteristics of HMT-BGS  of HMT 25-BGS and HMT 35-BGS are presented in Fig. L* and WI of the samples increased as treatment time and temperature decreased, ∆E of the starch samples increased with an increase in HMT treatment temperature and time , a* (4.19), b* (10.28) values, H° (81.20) and total colour difference (7.51) have also been reported for HMT potato starch with 20–25% at 120 °C44. Liestianty et al.45 showed colour characteristics of heat moisture treated sago starch at 28% moisture and 110 °C for 2–6 h. The authors observed that L*, a*, and b* values ranged from 80.58–82.17, 0.75–1.25 and 6.86–7.41, respectively. Nadir et al.44 reported a decrease in L* and b* values with an increase in a* value, but a notable reduction in Hue angle and colour difference after HMT. It can be observed that different HMT conditions have varying effects on colour properties. It has been reported that changes in colour characteristics of starches during physical methods like HMT could be due to the separation and purification of heterogeneous materials, including sugars, proteins, salt, and other elements44. Variations in colour are often attributed to the maillard reactions occurring between the amino acid groups in protein as well as the reducing sugars in starch46. Barua and Srivastav42 stated that the observed changes in colour may be due to caramelization reaction which produces simple sugars as a result of the breaking down of the starch molecules.Barua and SrivastavApplication of HMT to BGS is dependent on its functionality which can be deduced from characteristics such as WAC, OAC, SP and solubility of the starch. The functional properties of BGS treated under different HMT conditions are shown in Table 47. WAC indicates the water-binding and emulsifying potential of that specific starch47. WAC is a function of numerous strictures, including hydrogen bonds, degree of accessibility of water binding sites among the starches, conformational characteristics, stearic factors, and hydrophilic–hydrophobic balance49. Hoover and Sosulski50 showed that water-binding could be affected by the physical and chemical environment of starch suspensions, including pH, ionic strength, vapour pressure, temperature, and the presence or absence of a surfactant. Temperature greatly affects water absorption of starch because when the temperature rises, the molecules obtain sufficient thermal energy. The thermal energy weakens the intermolecular hydrogen bonds and increases interaction and affinity towards the water; as a result, the starch granules integrate with water51.The WAC and OAC of starch is the ability of starch to take up water or oil48. High swelling and solubility index are ascribed to the moveable granule structure, low molecular weight of amylose, amylopectin ratio and molecular features such as chain length, branching, micellar granular arrangement and the presence of lipids that form complexes52. Adebowale et al.53, in a study on the functional, physicochemical and retrogradation features of sword beans  acetylated and oxidized starch, observed that starch granules with a wide and strongly bonded micellar structure show a comparatively great resistance towards swelling, which may eventually result to a lesser solubility index.Swelling power is the hydration ability of starch at elevated temperatures. When starch is heated in water, the granules absorb water and swell; the attained thermal energy then weakens the inner bonds and binding forces of the starch granule. The swelling and solubility of the starches are directly influenced by the proportion of the crystalline and amorphous components of the starch granules49. Adebowale and Lawal54 reported that legume starches displayed improved functional properties than cereal and potato-based starches. It can be speculated that legume starches may attain a comparable degree of swelling and solubility as cereal starches at higher temperatures. The swelling power and solubility index indicates the extent of interactions between starch chains in the amorphous and crystalline region.The swelling power of starch from different legume sources is positively affected by the temperature and pH of the aqueous starch slurries. This is because thermal energy relaxes the strong intra-granular interactions, which disrupts the molecules of starch granulesThe highest values of OAC, SP and solubility of BGS with 15% MC were obtained at HMT 71.72 °C for 60 min, 80 °C for 30 min and 120 °C for 30 min respectively, whereas the highest values for WAC were obtained at HMT 100 °C for 17.57 min; 100 °C for 102.43 min; 80 °C for 90 min; and 120 °C for 90 min. Furthermore, the highest values of WAC, OAC, SP and solubility of BGS with 25% MC were obtained at HMT 100 °C 17.57 min and 80 °C for 30 min; 100 °C for 17.57 min and 100 °C for 102.43 min; 71.72 °C for 60 min and 100 °C for 60 min; as well as 120 °C for 30 min respectively. Consequently, the highest values of WAC, AOC, swelling power and solubility of BG starch with 35% MC were obtained at HMT 120 °C for 90 min and 128.28 °C for 60 min; 100 °C for 60 min and 71.72 °C for 60 min; 71.72 °C for 60 min and 80 °C for 30 min; as well as HMT 71.72 °C for 60 min respectively. Treatment time had a significant linear effect (p ≤ 0.05) on swelling power for HMT 15-BGS. In HMT 35-BGS, WAC was significantly affected by the quadratic effect of temperature and time, while solubility was significantly affected by the linear effect of time and the quadratic effect of temperature. However, temperature and treatment time had no significant (p ≥ 0.05) impact on the functional properties  of the HMT 25- BGS.64. The hydrophilic group of modified starches is more capable of binding with water and, therefore, facilitates water absorption39. The regression models for predicting the functional properties of heat moisture treated BGS is shown in Table A comparison of the functional properties of heat moisture treated starches from different sources, and the present study is shown in Table 65, Gunaratne and Hoover63, Senanayake et al.66,Olayinka et al.58, Zeng et al.64, Sharma et al.47 and Adebowale et al.60 all reported a decrease in swelling power and solubility for corn, cassava, sweet potato, peas, sorghum, waxy rice, pearl and finger millet starches respectively, due to the application of HMT. The decrease in solubility suggests a strengthening of bonds and an increase of interactions among amylopectin-amylopectin molecules, thereby slowing them down from leaching out of the starch granules. Consequently, the reduction in swelling power by HMT is caused by the internal reorganisation of starch granules, resulting in an interaction between starch functional groups, making it form a more ordered double-helical amylopectin side-chain cluster67. The decrease in swelling capacity can be attributed to the structural re-association of starch chains caused by the HMT and thus restriction for hydration, while starch solubility results from the leaching of amylose, which separates from starch granules and thus spreads out of starch granules during swelling68.Chung et al.55 reported increasing WAC and OAC in HMT buckwheat starch. Water absorption capacity may increase in the starch when amylose and amylopectin are loosely associated55. Nadir et al.44 also reported high WAC but a decrease in OAC by HMT in potato starch. This variation by HMT is due to changes in the hydrophobic and hydrophilic tendencies of the starch, which affects the oil and water absorption capacity of the starch61. The hydrophobic sites of starch promote oil absorption, while the hydrophilic site of the starch promotes water absorption69. Therefore, the ability of starch to absorb oil and water is a good indication of the emulsifying potentials of the starch60.Sarkar70. This phenomenon is significant in processing techniques, forming unique textural and structural characteristics in food products. Gelatinisation temperature also characterizes the starch type and is dependent on the glass transition of the amorphous fraction of the starch68. Cheng et al.70 postulated that the onset temperature (To) indicates the melting temperature of the weakest crystalline in starch granules, while the conclusion temperature (Tc) is the melting temperature of high perfection crystalline. Singh et al.14 stated that the gelatinization enthalpy (ΔH) primarily represents the loss of double-helical components within starch granules. The thermal properties (gelatinization) of BG starch under different HMT conditions are presented in Table Starch gelatinisation is an endothermic alteration significant for separating amylopectin double helices from the ordered structure to a disordered oneTo for BGS with 15% moisture content (MC) were obtained at HMT; 100 °C for 60 min while the highest values of Tp, Tc and ∆H were obtained at HMT 71.72 °C for 60 min. Similarly, the highest values of To and Tp of BGS with 25% MC were obtained at HMT 128.28 °C for 60 min, and the highest values of Tc and ∆H were obtained at HMT 120 °C for 90 min. Consequently, the highest values of To, Tp and Tc were obtained at HMT 71.72 °C for 60 min, while the highest ∆H values of BGS with 35% MC were obtained at HMT 100 °C for 60 min (Table To), (75.33 °C) at the peak temperature (Tp), (79.17 °C) at the gelatinization conclusion temperature (Tc) and 11.73 (J/g) for enthalpy of gelatinization (ΔH)72. The higher gelatinization temperature for BGS may result from the more rigid granular starch structure14. This is associated with structural variations within the starch granules involving amylose-amylose and amylose–lipid interactions. These interactions reduce the mobility of the amorphous region, which result in higher temperature required for the swelling and disruption of the crystalline regions71.The highest values of 63. Furthermore, these differences in gelatinization temperatures could be due to the amylose content, size, and the re-alignment of starch helices within the starch38. In HMT 15-BGS, the To, Tp and Tc of the samples decreased as treatment time and temperature increased  of the starch samples decreased with an increase in HMT treatment temperature and time  effect on the thermal properties of the heat moisture treated BGS. The regression models for predicting thermal properties of heat moisture treated BGS  are shown in Table 75 postulated that the extent to which amylose-amylose, amylose-amylopectin, and amylose–lipid interactions associate during HMT is influenced by the starch source amylose chain length and the moisture content during HMT. An increase in gelatinization parameters on HMT has been linked to the interaction between amylose–amylose, amylose–amylopectin, amylopectin–amylopectin chains, and the formation of other complexes between starch amylose and lipids76. Moreover, an increase in gelatinization parameters could suggest that crystallites disrupted by HMT may have combined to form larger ones. However, Zavereze et al.68 indicated that the strength of intermolecular bonds in starch promoted by HMT requires a higher temperature to gelatinize the starch granules. Zeng et al.64 and Sharma et al.47 reported an increase in To, Tp, and Tc with a decrease in ∆H during HMT of waxy rice and pearl millet starch. A reduction in ∆H after HMT has also been reported for pea, navy bean, regular maize starch, lentils, and waxy potato starches77. Similarly, reduced ∆H promoted by HMT were reported in jack bean, corn, potato, and cassava starches78. The reduction in gelatinization enthalpy by HMT results from the disruption of the crystallites, which are unstable, hence leading to a lower degree of crystallinity and, therefore, requiring less energy for disruption63. Chung et al.62 stated that reduction by the HMT demonstrates that the high temperature during HMT may increase the mobility of double helices forming the crystalline structure, thereby disrupting the hydrogen bonds between the helices. Hormdok and Noomhorm79 showed that the reduction in gelatinization after HMT may be due to the partial gelatinization of amylose and amylopectin molecules, which becomes highly unstable during heating. Cheng et al.70 reported that HMT at 25% moisture had a significant effect on gelatinization parameters of maize starch. However, the findings of this present study negate the observation of Cheng et al.70. This may be due to the differences in HMT conditions used in Cheng et al.70  and starch origin used in the present study.HooverL*; ∆E and WI; ∆E and L*; H° and a*; To and Tp; Tp and Tc and To and Tc. However, there was no correlation between the thermal properties and colour characteristics except for To, which had a significant correlation with b*. Furthermore, it could be observed that WAC correlated with To and Tp. A correlation between WI and L*; ∆E and WI; ∆E and L*; H° and a*, To and Tp; Tc and Tp; OAC and Tc was observed under HMT 25. However, there was no correlation between the thermal properties and colour characteristics , unmodified and HMT 15 showed lower peak intensities than HMT 25 and 35 BG starches. The variations in peak intensities could be related to the difference in amylose composition85. Other peaks were observed around 1650 cm−1 wavelength, which is probably associated with bending vibrations of H2O absorbed in the amorphous regions of the starch64. Similar observations were reported for potato, corn and wheat starches85.FTIR has been suggested to be sensitive to changes in structure on a molecular level (short-range order). Furthermore, it provides information on the structural arrangement of starch chains near the granule surface because the infrared beam penetrates only to a certain extent into the granuleL* values are desired in processed food, as these parameters can influence consumer acceptability. The enhanced oil absorption in (302.1) and reduced water absorption (162.9) of the HMT35-BG starch show desirable attributes in food production where starch is applied as a thickener. This is because of its ability to form firm gels, an advantage required in confectionaries and baked products as well as in the manufacture of noodles with greatly enhanced textural properties. Treatment time had a significant linear effect (p ≤ 0.05) on swelling power for HMT 15-BG starch. HMT 25 and HMT 35 both had the highest desirability value of 1.00 each. This result implies that processing BG starch at 96.77 °C & 104.16 min (HMT25) and 76.55 °C 81.65 min (HMT35) yielded optimal colour, functional and thermal properties. Information obtained from scanning electron micrograph indicates oval and round shapes for BGS granules, with varying sizes. The range of the granule size width was 4.2–4.7 mm and 10 μm for length. The unmodified starch presented a more agglomerated and cracked surface than the heat moisture-treated starches. The HMT starches seemed to have more minor cracks and a smoother surface was formed as HMT conditions (moisture and temperature) increased. Unmodified and heat moisture treated BGS showed characteristic FTIR bands linked with common starches. All the samples displayed complex vibrations in the region below 1000 cm−1 due to the skeletal vibrations of the glucose pyranose ring. The findings of this study will help food processors to tailor the process conditions of heat moisture treated BGS for industrial application and promote the diversified use of BG in the development of confectionaries, baked food products and noodles with enhanced texture properties.Heat-moisture treatment had a slight effect on the colour, functional and thermal properties of BGS. Reduced colour change and high Supplementary Figures.


Considering the increasing number of adults seeking orthodontic treatment, and the possible need for bracket bonding to monolithic zirconia restorations, knowledge about the preferred type of bracket  and the most efficient surface treatment is imperative to achieve acceptable shear bond strength (SBS). This study aimed to assess the effect of different surface treatments and orthodontic bracket types on SBS of high-translucent zirconia. n = 31) for the following surface treatments: no surface treatment (control group), airborne-particle abrasion (APA), tribochemical silica coating with CoJet, and CO2 laser irradiation. The mean surface roughness  was measured. The SBS was measured after bracket bonding and thermocycling. Data were analyzed by two-way ANOVA, Tukey, Kruskal–Wallis, Mann–Whitney, and Fisher exact tests, and Bonferroni correction (α=0.05).  Totally, 248 disc-shaped zirconia specimens were assigned to two groups for bonding to metal and ceramic brackets. Each group was divided into four subgroups (P < 0.001). The APA and CoJet subgroups were not significantly different regarding Ra (P=0.754). All other pairwise comparisons yielded significant differences (P < 0.001). Bracket type, surface treatment, and their interaction significantly affected the SBS (P < 0.001). Ceramic brackets bonded to zirconia surfaces treated with CoJet yielded the maximum SBS while ceramic brackets bonded to control and lased surfaces resulted in minimum SBS. No significant difference was noted in the SBS of different surface treatment groups when metal brackets were used (P > 0.05).  The mean Ra value was significantly different among the surface treatment subgroups ( The use of ceramic brackets and CoJet surface treatment would be the most appropriate combination to achieve optimal bonding to high-translucent zirconia restorations. The demand for orthodontic treatment is on the rise. In the past, children and young adults were the main candidates for orthodontic treatment; however, an increasing number of adults currently seek orthodontic treatment . In thisBracket bonding is a critical step in orthodontic treatment . The sur2), erbium-doped yttrium aluminum garnet, or femtosecond laser irradiation [2 laser treatment of ceramic surfaces for bonding to composite resin or resin cements. Controversy still exists regarding an ideal zirconia surface treatment to provide adequately high bond strength and minimize the risk of bracket debonding [Hydrofluoric acid cannot be used for the surface treatment of zirconia since zirconia is devoid of a glass phase . The sugadiation . Of diffadiation . Nonetheadiation  in a metebonding . Since tebonding , their sIn recent years, the demand for invisible orthodontic treatments with the minimal show has increased, which led to the development of ceramic brackets. Considering the differences in properties of ceramic and conventional metal brackets, it is important to find an efficient bonding protocol for them. Information on this topic is limited, and controversy exists regarding the preference for using ceramic brackets on zirconia restoration surfaces , 4, whic2 laser irradiation. To calculate the required sample size, the appropriate effect size had to be considered. Thus, two-way ANOVA feature of GPower 3.1.9.2 software was used for this purpose. Accordingly, the sample size was calculated to be 29 in each group, considering the effect size of 0.25 (medium effect size), study power of 0.90, alpha-0.05, presence of 8 groups, and degree of freedom of 3 . To increase the accuracy of the results, the sample size in each group was increased to 31. Thus, in this in vitro experimental study, 248 disc-shaped specimens with 10 mm diameter and 3 mm thickness were fabricated from monolithic super-translucent and polychromatic yttrium-stabilized tetragonal zirconia (Y-TZP) ceramic  by a milling machine . The specimens were sintered at 1450°C according to the manufacturer's instructions and were then polished with 600, 800, and 1200-grit silicon carbide abrasive papers for 15 seconds under water coolant [In this study, eight groups were required to measure the SBS of metal and ceramic brackets to zirconia with four surface treatments of control, sandblasting, CoJet, and COn = 31) for the following surface treatments  and was then subjected to CO2 laser irradiation  at 10600 nm wavelength, 4 W power, 159.22 mJ energy density, 50-second irradiation time, and 4 mm focal spot in continuous-wave mode [The specimens were cleaned in an ultrasonic bath (96% isopropanol for 3 minutes at room temperature) and dried. They were then assigned to two groups for bonding to metal and ceramic brackets. The specimens in each group were divided into four subgroups  was used to assess the surface roughness of specimens after surface treatments. The mean Ra parameter was measured for this purpose. The Ra value was measured at the respective area three times for each specimen with a stylus speed of 0.1 mm/second with 0.001 µm accuracy, and the mean Ra value was calculated for each specimen .A qualitative assessment of the surface was performed by using a scanning electron microscope (SEM). One additional specimen was fabricated for each study group to qualitatively assess the morphological surface changes after each surface treatment under the SEM . Micrographs were obtained at ×2000 magnification.2 .The specimens were cleaned again in an ultrasonic bath (96% isopropanol for 3 minutes at room temperature), rinsed with water, and air-dried. The bonding procedures were performed for the metal and ceramic bracket groups according to the manufacturers' instructions. A thin uniform layer of silane primer containing MDP  was applied on the surface by a disposable microbrush as instructed by the manufacturer and dried with gentle air spray. Metal  and ceramic  mandibular central incisor brackets were then bonded to the prepared surfaces by a resin cement . Excess cement was removed by the sharp tip of an explorer, and the resin cement was light-cured at each side of the bracket for 20 seconds with a light intensity of 1400 mW/cm2).The zirconia bracket assembly was stored in distilled water at 37°C for 24 hours and then underwent 10,000 thermal cycles between 5 and 55°C with a dwell time of 30 seconds and a transfer time of 20 seconds . The SBS  Score 0: no adhesive remaining on the ceramic surface  Score 1: less than 50% of adhesive remaining on the ceramic surface  Score 2: more than 50% of adhesive remaining on the ceramic surface  Score 3: the entire adhesive remaining on the ceramic surface with the bracket mesh impression on itThe mode of failure was determined under a video measuring machine . Both bracket and zirconia bonding surfaces were inspected. The adhesive remnant index (ARI) score was calculated as follows to classify the failure mode: .  Score Also, the failure mode was categorized into cohesive , adhesive (debonding at the resin cement-bracket or resin cement-zirconia interface), and mixed (a combination of adhesive and cohesive failures) types .The normality of data distribution was analyzed by the Shapiro–Wilk test. The homogeneity of variances was analyzed by Levene's test. Since the assumption of homogeneity of variances was not met, the partial eta squared was used to homogenize the variances. The SBS data were analyzed by two-way ANOVA, which was followed by pairwise comparisons with the Tukey test. Since the assumptions were not met for the surface roughness data, the surface roughness of the groups was compared by the Kruskal–Wallis test followed by the Mann–Whitney test with Bonferroni correction for pairwise comparisons. The Fisher exact test was applied to compare the frequency of modes of failure in the groups. All statistical analyses were carried out using SPSS version 25  at a 0.05 level of significance.P < 0.001). Pairwise comparisons found no significant difference between APA and CoJet subgroups (P=0.754). However, other pairwise comparisons revealed significant differences (P < 0.001). The maximum surface roughness value was recorded for specimens subjected to APA and CoJet followed by CO2 laser and control groups.SEM assessment of surface changes following different treatments at ×2000 magnification revealed that the polished surface of the control specimen was smoother than other surfaces, and had more superficial vertical and horizontal scratches with slight prominences and depressions. The lased surface had nonhomogeneous and nonuniform scratches. Also, some areas had deep grooves, agglomerates, and a nonhomogeneous surface. Airborne-particle abraded and CoJet-treated surfaces had a wrinkled appearance such that cavitated and porous scratches were noted on the entire surface. These surfaces were much rougher and more heterogeneous than other surfaces. Also, agglomerated and wrinkled areas were seen all over the surface .P < 0.001). Assessment of the effect of bracket type, irrespective of surface treatment type, showed that metal brackets yielded a significantly higher bond strength to zirconia than ceramic brackets. Assessment of the effect of surface treatment, irrespective of bracket type, indicated no significant difference in SBS of APA and CoJet subgroups (P=0.169). However, other subgroups had significant differences in SBS (P < 0.001). The maximum SBS was noted in CoJet and APA subgroups followed by CO2 laser and then the control subgroup.Two-way ANOVA was applied to assess the effect of bracket type and different surface treatments on SBS . The resP=0.999), CoJet-treated (P=0.268), or lased (P=0.213) surfaces. No significant difference was found between metal brackets bonded to lased surfaces and metal brackets bonded to airborne-particle abraded (P=0.270) and CoJet-treated (P=0.999) surfaces, or metal brackets bonded to CoJet-treated surfaces and metal brackets bonded to airborne-particle abraded surfaces (P=0.332). No other significant differences were noted in pairwise comparisons (P > 0.05). The maximum SBS was found in ceramic brackets bonded to CoJet-treated surfaces while the minimum SBS was recorded in ceramic brackets bonded to control surfaces . Compared with the groups with ARI score 1 in 100% of the specimens, 29% and 39% of metal bracket/CoJet and ceramic bracket/CoJet specimens showed ARI score 2, respectively, and 23% of ceramic bracket/control specimens showed ARI score 0. Except for the ceramic bracket/control group, in which 23% of specimens showed adhesive failure at the resin cement-zirconia interface, other failures were mixed. No cohesive failure was noted in zirconia specimens or brackets.Considering the optimal properties of zirconia, it is commonly used as a prosthetic restorative material.  Thus, orThermocycling is a type of artificial aging to simulate clinical conditions, which evaluates the resistance of the bond to hydrolysis , 22. In Evidence shows that the required bond strength value between orthodontic brackets and the underlying substrate should be 6–8 MPa . The meaAccording to the present results, the type of surface treatment significantly affected the SBS of orthodontic brackets to zirconia, which was in agreement with previous findings , 25, 27.2 laser and control subgroups; these results were also confirmed by SEM assessment of surfaces and agreed with the results regarding the effect of surface treatment on SBS. However, these findings contradicted the results regarding the interaction effect of bracket type and surface treatment on SBS, which may indicate that the SBS does not merely depend on surface roughness created by different mechanical surface treatments, but other factors such as the bracket type also affect it.The present study also evaluated the effect of different surface treatments on the zirconia surface quantitatively while very few of the studies on bond strength of orthodontic brackets to zirconia measured the surface roughness of specimens as well. The maximum surface roughness was recorded in the APA and CoJet subgroups followed by CO2 laser treatments of zirconia surfaces yielded higher bond strength to ceramic brackets than the control specimens. Higher bond strength in the CoJet compared with the APA subgroup, despite equal surface roughness values, may be due to the use of a primer containing silane and MDP [CoJet, APA, and CO and MDP , 28. How and MDP  found no and MDP , and notMetal brackets had a significantly stronger bond to airborne-particle abraded, CoJet-treated, and lased surfaces than the control specimens in the present study. This finding was in contrast to the results of some studies , 20, 27 The result of the comparison of SBS of metal and ceramic brackets in the present study depends on the selected surface treatment, such that ceramic brackets yielded a higher SBS than metal brackets when bonded to CoJet-treated surfaces, compared with other methods. However, the SBS of ceramic brackets to airborne-particle abraded surfaces was comparable to the SBS of metal brackets bonded to surfaces treated with other methods. The SBS of ceramic brackets bonded to lased surfaces was lower than that of metal brackets. The available studies on SBS of metal and ceramic brackets to zirconia surfaces treated with different methods have reported controversial results, which are also different from the present findings. The results reported in such studies did not depend on the type of surface treatment used. Sanz et al.  reportedThe magnitude and direction of loads applied to zirconia during orthodontic bracket debonding are different from the commonly applied loads to these restorations . Since tIn brief, the present results may indicate that in presence of zirconia restorations, the bracket material must be carefully selected and a zirconia surface treatment compatible with the bracket type should be chosen to achieve optimally high bond strength. The use of MDP-containing primers should also be carefully considered . NonetheIn vitro design was another limitation of this study, since the complex oral conditions cannot be perfectly simulated in vitro. Additionally, some unexplored factors, such as fluid  or fluorWithin the limitations of this in vitro study, it may be concluded that the SBS of orthodontic brackets to HT zirconia depends on the type of bracket and surface treatment. Of the groups evaluated in this study, ceramic brackets bonded to CoJet-treated zirconia surfaces yielded the maximum SBS. Nonetheless, no significant difference was noted in the SBS of different surface treatment groups when metal brackets were used.


The surface characteristics of the LD layer are influenced by the etching time and affect the SBS of the bond of zirconia to resin cement. HF etching for 90–120 s after GCSD results in zirconia with SBS and bond durability comparable to LD.Glass-ceramic spray deposition (GCSD) is a novel technique for coating lithium disilicate (LD) glass-ceramics onto zirconia through simple tempering steps. GCSD has been proven to improve the bonding of zirconia to resin cement, but the effect of etching time on GCSD and the long-term durability of the bond achieved remain unknown. The effects of air abrasion with aluminum particles (ABB) and air abrasion (GAB) or etching with 5.0% hydrogen fluoride (HF) for 20, 60, 90, and 120 s  on the resin cement–zirconia bond were studied. LD was included as a control (LDG). The microstructure, sub-micron roughness, wettability, and phase changes of samples were analyzed. After resin cement was bonded to zirconia, half of the samples were subjected to thermocycling (5000 cycles at 5–55 °C). The bond strengths of the samples were determined in shear bond strength (SBS) tests (n = 10 per group). An LD structure can be formed on zirconia after GCSD and proper etching processes, which result in high roughness and a hydrophilic nature. GCSD and HF etching significantly improved SBS, with G90 and G120 samples with pre- or post-thermocycling exhibiting SBS values comparable to those of LDG ( Zirconia restorations have attracted the attention of clinical dentistry because of their excellent mechanical properties, high transparency, and natural appearance ,2,3. TraSeveral methods are commonly used to improve the bonding of zirconia in clinical dentistry. Air abrasion using alumina oxide particles improves micromechanical interlocking at the zirconia surface, which can improve bonding ,16,18. SThe gold standard for surface treatment of zirconia is to combine air abrasion and an MDP-based primer to increase the micromechanical interlocking and chemical bonding of zirconia to dental resin. However, how to achieve bond strength and durability that are comparable to those achievable with traditional dental ceramics remains unclear. Glass-ceramic spray deposition (GCSD) is a surface treatment technique that can improve the bond effectiveness of zirconia restorations . GCSD inThe GCSD technique has been proven to improve the bond strength between zirconia and resin cement, but the effects of etching time on the bond strength and durability after GCSD treatment are not yet fully understood . One objThe microstructure of the testing samples is presented in p < 0.001). No statistically significant differences were detected between groups with different etching times (p > 0.363), between LDG and ABB (p = 0.301), or between LDG and GAB (p = 0.516).2Si2O5) crystallization (JCPD #14-0322). For ABB, broadened and shifted peaks of tetragonal zirconia , monoclinic zirconia , and m/t-ZrO2 were observed . For GAB, the main reflecting peak was at 31.5° of t-ZrO2, and diffraction peaks of m-ZrO2 were also present, with diffraction peaks at 28.2°, 34.3°, and 35.3° corresponding to −1 1 1, 0 0 2/0 2 0, and 2 0 0/0 0 2, respectively. Notably, the glass-ceramic coating on the Y-TZP surface transformed from an amorphous phase to Li2Si2O5. Hence, a reflecting peak for Li2Si2O5 (LS) was also detected. For the G20, G60, G90, and G120 groups, the reflection peaks of LS become more significant with increasing etching time, and the reflecting peak of t-ZrO2 also became more distinct.p > 0.999) and G90 (p = 0.269) groups exhibited no statistically significant differences from the G120 group. After 5000 thermocycles, the LDG and G120 groups exhibited significantly higher SBS than the AAB, GAB, G20, and G60 groups (p ≤ 0.001). The LDG, G90, and G120 groups were comparable in terms of SBS (p > 0.760). All groups exhibited significant SBS reduction after thermocycling (p < 0.001). The LDG, AAB, and G120 groups exhibited better bonding durability. All groups that did not undergo thermocycling exhibited mixture failure predominantly, except AAB, for which 50% of the failures were classified as adhesive failures. The G90 and G120 groups exhibited 100% mixture failures. After 5000 thermocycles, the failure mode was predominantly mixture failure, except for the AAB and G20 groups, for which it was predominantly adhesive failure. Note that the GAB and G120 groups exhibited 100% mixture failures. The GCSD technique was developed for creating thin and dense LD glass-ceramic coatings on zirconia surfaces to overcome the inadequacy of the bonding performance of zirconia without changing the phase structures or physicochemical properties of zirconia. Sundfeld et al.  explainet-ZrO2  becomes clear because of the level of etching. The microstructure of the four GCSD groups  comprises LD crystals exhibiting a long-needle shape embedded in a glassy matrix, and the degree of crystallinity increases with increasing etching time  on SBS. Results indicate that the longer the etching agent stays on the zirconia surface, the more deeply the glassy phase is eroded and the more obviously the surface topography changes. From the change in the surface phase structure , the chaing time . These ring time  indicateing time , making ing time , with thing time , faciliting time . Thus, tt-m) phase transformation, which would decrease the fracture strength, surface damage, and microcrack formation. The current study examined air abrasion for one group (AAB), and the experimental results were consistent with those reported in the literature (t-ZrO2 (1 1 1) was observed after air abrasion. Air abrasion after GCSD treatment (GAB) results in the appearance of multi-characteristic peaks such as m-ZrO2, t-ZrO2, and LS. However, the prominent peak was zirconia because of the LD glass-ceramic coating being destroyed by Al2O3 particle abrasion and then debonding, so only a tiny amount of LD glass-ceramic remained on the surface . Thus, air abrasion after GCSD treatment is not recommended.Air abrasion makes the zirconia surface disordered and irregular, which facilitates interlocking between the resin cement and zirconia and increases the SBS. Skienhe et al.  suggesteterature . A broad surface . The LD n cement . Howeverp < 0.01)  . These r < 0.01) . This ca < 0.01) , indicatp < 0.01), consistent with previous studies [Thermal cycling was used as an artificial aging method to evaluate bond durability in the current research. All groups exhibited significant SBS reductions  after ar studies ,23,28. T studies . CompareNote that the LDG group exhibited the lowest reduction (43.9%), which might be because of its higher SFE and lower Ra, making it difficult for water molecules to enter the interface of the material. However, the precise reasons still need to be examined in the future. Based on the results of this study, 5% HF etching for 90 to 120 s after GCSD treatment is recommended to achieve the strongest and most durable bond between resin cement and zirconia.2 area. Contact angles (CAs) were measured to investigate the wettability of the samples using a CA analyzer . Each reported CA value was calculated based on ten independent attempts. The surface free energy (SFE) was calculated by the Girifalco–Good–Fowkes–Young model [The surface morphologies of the samples were assessed using a field emission scanning electron microscope  in secondary electron mode. Each sample’s sub-micron roughness (Ra) was analyzed via atomic force microscopy  over a 10 × 10 μmng model , based oZ-Prime Plus  was used to prime the AAB group, and Monobond N  was used to prime the other six groups. A plastic tube (6.0 mm in diameter and 2.0 mm in height) was then attached to each sample with a piece of double-sided polyethylene adhesive tape to define the bonding area. Resin cement  was flowed into the plastic tube and light-cured for 10 s , and any residual cement was removed. One hour was allowed to elapse to ensure that the cementation was complete. One-half of the samples were immersed in distilled water at 37 °C for 24 h. The remaining samples were placed in a self-assembling thermocycling apparatus and cycled between 5 and 55 °C in distilled water for 5000 cycles with a dwell time of 30 s and a transfer time of 15 s, in accordance with the ISO 10477 standard . A univep < 0.05 unless otherwise specified.The sample size was calculated based on a power analysis with a power of 1.0 and an α error of 0.05, which enabled clinically justified recommendations. Normality was assessed primarily by the Shapiro–Wilk test. Statistical comparisons of the test results for the various groups were performed using a one-way analysis of variance (ANOVA) with Tukey’s honestly significant difference (HSD) tests for multiple comparisons. The statistical analyses were performed using SPSS 19  and Prism 9.0 . The threshold for significant differences was set to This study aimed at investigating the effect of etching on GCSD treatment effectiveness and at evaluating whether zirconia can achieve a sufficiently durable bond with resin cement after GCSD treatment. The acid etching time significantly affects the microstructure, sub-micron roughness, and wettability of the LD layer, which improves the SBS. Particularly for the G90 and G120 groups, SBS values comparable to those for LD were achieved even after artificial aging by thermal cycling. GCSD accompanied by 5% hydrogen fluoride acid etching for 90–120 s and appropriate chemical priming is recommended to produce the best surface physicochemical structure to enhance the bonding performance between zirconia and resin cement, expanding the usable range and prolonging the life of zirconium-based dental restorations.


Subsequently, the coating was subjected to ultraviolet (UV) light to enhance the biological inertness of zirconia. Scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD) and contact angle analysis were conducted to confirm the surface characteristics. Afterwards, in vitro assessments of cell adhesion, proliferation and osteogenic differentiation of MC3T3-E1 cells were performed. Zirconia samples were implanted into rat femurs to assess biocompatibility and host tissue response in vivo. Micro-CT evaluation and histological testing were conducted. After UV irradiation, the content of hydroxyl groups and hydrophilicity of TiO2-modified zirconia were significantly increased. The results of in vitro experiments showed that TiO2-modified zirconia subjected to UV light could promote cell proliferation and spreading, enhance ALP activity and the degree of mineralization, and upregulate osteogenesis-related genes. Furthermore, in vivo assessments confirmed that UV-irradiated TiO2-modified zirconia implants maximized the promotion of osseointegration. TiO2-modified zirconia after UV treatment will have broad clinical application prospects in improving the osseointegration of zirconia implants.Zirconia is a superior implant material owing to its high mechanical strength, durable corrosion resistance, superior aesthetic effect and excellent biocompatibility. However, the bioactivity of zirconia surfaces remains a great challenge for implant osseointegration. A titania (TiO Dental implantation is the primary treatment option for patients suffering from tooth loss or defects . AlthougSeveral strategies have been employed to enhance the bioactivity of zirconia , such as2 immediately after they are exposed to oxygen or the atmosphere . Group C required no additional treatment. The TiZ group specimens were placed into 1 mol/L ZrOCl2 and 0.5 mol/L TiO2 mixed suspensions and subsequently heated in a 95°C water bath for 4 h. To prevent evaporation of the liquid, the beaker was covered with a lid. Eventually, the zirconia samples of each group were densely sintered in a furnace  at a temperature of 1530°C. After dense sintering, half of the TiZ group samples were subjected to UV light  by a UV irradiation machine  and were classified as the UV-TiZ group. The UV-TiZ group samples were placed 10 cm below the lamp tube and irradiated for 15 min. In addition, each group of rod zirconia implants  were prepared in the same way as zirconia discs.Pre-sintered zirconia discs  with a diameter of 14 mm and a thickness of 2 mm were gradient polished to 1200 mesh and ultrasonically cleaned with distilled water. All zirconia specimens were randomly divided into 2 groups: the control group (C) and the TiOn = 4) was evaluated by a contact angle goniometer . The bonding strength of the titania coating and zirconia (n = 5) was measured by using a scratch tester . A rockwell head with a radius of curvature of 200 μm was then used with a loading rate of 20 N/min and a terminal load of approximately 100 N. The critical load (Lc) value of the coating is obtained by signal acquisition.The surface morphology of each group of zirconia specimens was observed by SEM . The samples were subjected to XPS  to evaluate the changes in the chemical state of oxygen on the surface of the zirconia discs after UV treatment. Crystal phase analysis of all samples was performed by XRD examination . The water contact angle of the specimen  were cultured to evaluate the cytocompatibility of each zirconia group. Under standard conditions , cells were cultured in a formulated alpha minimum essential medium containing 10% fetal bovine serum and 1% penicillin. The medium was refreshed every 48 h.4 cells/well in a 24-well plate. At each culture period (1 and 3 d), a live/dead staining kit  was utilized to assess the cytotoxicity of zirconia specimens. Calcein-AM was a staining reagent for fluorescently labelling living cells with green fluorescence, and its working concentration was 1 μΜ. In addition, PI (3 μΜ) only stained dead cells and excited red fluorescence. Finally, dyed cells were visualized through fluorescence microscope .MC3T3-E1 cells were seeded at a density of 1× 104 cells/well in 24-well plates (n = 4). After 1, 3 and 5 d of culture, media containing 10% CCK-8 reagent was added to the wells as directed and incubated at 37°C for 2.5 h. Finally, 100 μL of solution was removed from each well into a 96-well plate, and the absorbance was read at 450 nm.A CCK-8 kit  was used to detect the proliferation of MC3T3-E1 cells cultured on the surface of groups C, TiZ and UV-TiZ. The seeding density was 2.5×104 cells/well) were seeded on zirconia discs in a 24-well plate. After culturing for 1 and 3 d, the cells were fixed with 4% paraformaldehyde for 30 min and infiltrated with 0.1% Triton X-100 for 10 min. Thereafter, F-actin and nuclei were stained with phalloidin  and DAPI , respectively. A fluorescence microscope  was utilized to observe the morphology of the cells cultured on zirconia discs.To visualize the cytoskeleton, MC3T3-E1 cells . Finally, all samples were observed after gold spraying.In addition, cell morphology was observed by SEM . MC3T3-E1 cells were seeded in a 24-well plate at a density of 1×104 cells/well (n = 4). After culturing for 24 h, the medium was replaced with fresh osteogenic induction medium containing dexamethasone , β-glycerophosphate  and L-ascorbic acid . After 4 and 7 d of osteogenic induction, the total amount of protein was examined by a BCA protein assay kit . An ALP assay kit  was employed for the quantitative assessment of ALP. Finally, the ALP activity was measured according to the instructions, and it was standardized to the total protein content. Afterwards, at testing time points on 4 and 7 d of induction, the cells were stained with a BCIP/NBT Kit  and observed by stereomicroscopy .MC3T3-E1 cells were incubated in a 24-well plate at a density of 2×10n = 4) at a density of 1×105 cells/well. After 7 and 14 d of osteogenic induction, the cultured cells were dyed with the prepared 0.2% Alizarin Red staining solution  and observed by microscopy . Finally, the mineralized nodules were dissolved in 10% cetylpyridinium chloride , and the absorbance was read at 620 nm.ARS and its quantitative results were employed to assess the degree of mineralization of MC3T3-E1 cells seeded on each group of zirconia (5 cells/well on the zirconia disc in each well (n = 4). After 7 d of osteogenic induction, the expression levels of osteogenic genes, including ALP, runt-related transcription factor 2 (Runx2), collagen-I (COL-I), osteocalcin (OCN) and osteopontin (OPN), were detected. Total RNA was isolated by the TRIzol  method, and the concentration was determined by Nanodrop . The extracted RNA was prepared into cDNA by employing the Reverse Transcription Takara kit . Finally, quantitative RT–PCR was performed with SYBR Green chemistry . Primers for osteogenesis-related genes are listed in MC3T3-E1 cells were seeded in a 24-well plate at a density of 1×10All animal experiments were approved by the ethics committee of Beijing Stomatological Hospital affiliated with Capital Medical University and complied with the “Guide for the Care and Use of Laboratory Animals”. A total of 78 Eight-week-old male Sprague Dawley (SD) rats were randomly divided into 3 groups: C, TiZ and UV-TiZ. After general anaesthesia by intraperitoneal injection of chloral hydrate, the legs of each rat were shaved and sterilized. An incision was made in the rat’s leg to expose the femur. A bone defect of 1 mm in diameter and 10 mm in length was prepared and cooled with saline to prevent osteonecrosis. Afterwards, each group of zirconia implants prepared according to the method described in 2.1 were placed into the cavity, and the wound was carefully sutured. After 4 and 8 w of healing, the rats were sacrificed. The implant-containing femur specimens were fixed with 4% paraformaldehyde for further evaluation.High-resolution micro-CT  was employed to scan the obtained specimens. The scanning parameters were set as follows: the spatial resolution was 18 μm . Three-dimensional (3D) images of each group of samples were reconstructed by CTvox  software. Reconstructed data were further analyzed by CT-Analyzer through controlling the minimum grey threshold value of 30 and the maximum of 255. The region of interest (ROI) was determined as a column (1.5 mm in diameter) from the centre of the implant and 1.0 mm above the epiphyseal growth layer line.50 axial images were reconstructed into a 3D image which was used to measure the bone parameters. The important bone parameters, including new bone volume over total bone volume (BV/TV), mean trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular separation (Tb.Sp), were calculated (n = 4).n = 5) were carefully trimmed to expose the top of the implant. Thereafter, the cut bone tissue was fixed on the jig and clamped in the same position on the different implants. The implant was pulled out at a speed of 1 mm/min, and the maximum force was recorded.The integration of the implant and bone tissue was tested by a universal testing machine . The femoral samples (n = 4).After the SD rats were sacrificed, the bone fragments were fixed with 4% paraformaldehyde for 48 h. Subsequently, the bone blocks were soaked in graded ethanol solutions (60 increased to 100 v/v%). After dehydration, the tissue blocks were placed in plastic moulds and embedded in an EXAKT 520 light-curing embedding machine using Technovit 7200 resin . The diamond blade cut the specimen along the long axis of the implant to obtain a 200-μm-thick tissue piece. Thereafter, the sections were graded and polished to 20 μm. Finally, the samples were stained with methylene blue and fuchsin staining reagent and observed by microscopy . The percentage of bone-implant contact (BIC%) was measured by image pro plus software , was used to determine the sample size. Based on the data from our pilot study, the sample size was identified to provide statistically significant measure of the various tests used in this study .All statistical data are expressed as the means with standard deviations. LSD analysis was used to compare pairs after one-way analysis of variance (ANOVA). Statistical significance was defined as *2 coating was prepared on the surface of zirconia. 2 coatings were rough.2 was prepared on the surface of zirconia, and crystal phase transformation did not occur after UV irradiation (p < 0.05). The contact angle of zirconia (81.35°) was significantly higher than that of the TiO2 coating (43.95°). Moreover, after UV irradiation, the hydrophilicity of the TiO2 coating was significantly improved to 9.33°. The excellent adhesive strength of the coating plays a key role in its mechanical properties. The maximum force on the titania coating reached (20.3 ± 1.2) N. The titania coating had a high enough bond strength to withstand the load.The high-resolution O1s peaks of the XPS spectrum of the TiZ and UV-TiZ groups could be separated into two peaks at approximately 530.0 and 531.1 eV , and theadiation . The conAfter live/dead cell staining, live cells were dyed green with calcein-AM, and dead cells presented red due to propidium iodide. p < 0.05).The SEM images of MC3T3-E1 cells seeded on different groups were displayed in p < 0.05).p < 0.05).After 7 and 14 d of osteogenic induction, MC3T3-E1 cells seeded on the surface of the UV-TiZ group exhibited the most mineralized nodules and the highest mineralization level shown in . Furtherp < 0.05). The degree of gene expression upregulation showed a trend as follows: UV-TiZ > TiZ > C. In addition, the differences between the groups were significant (p < 0.05).The RT–PCR analysis of ALP, Runx2, COL-І, OPN and OCN gene expression is shown in Micro-CT was employed to evaluate the new bone formation around different implants after 4 and 8 w of healing, and the results are presented in p < 0.05), and that of the TiZ group was also significantly higher than that of group C (p < 0.05).Methylene blue and fuchsin staining was employed to assess the osseointegration of the implants in each group. 2-coated modified zirconia. The preparation of TiO2 coating took advantage of the hydrolysis of zirconium oxychloride and the large porosity of pre-sintered zirconia. Consequently, TiO2 coatings were strongly bonded to the substrate without damaging the mechanical strength of the zirconia (2 coating-modified zirconia, UV treatment was introduced to achieve a more ideal osseointegration effect.After extensive long-term research, zirconia has been confirmed to be suitable for application in dental implants . A numbezirconia . To enha2-modified zirconia surface after UV irradiation, which caused an increase in hydrophilicity. These physicochemical changes were mainly related to UV-induced direct photolysis of hydrocarbons and reactive oxygen species generation on the TiO2 surface. Under ultraviolet light irradiation, electrons in the valence band of TiO2 are excited to the conduction band, electrons and holes migrate to the surface of TiO2 and electron-hole pairs are generated on the surface. The electrons react with Ti, and the holes react with the surface bridge oxygen ions, forming respectively positive trivalent titanium ions and oxygen vacancies. In turn, the oxygen and water in the environment were converted into various free radicals, including superoxide and hydroxyl groups, to increase the polarity and finally resulted in an increase in hydrophilicity (The successful loading of dental implants is determined by their physicochemical surface properties. The XPS results demonstrated that a large number of Ti-OH bonds were excited on TiOhilicity . Hydrophhilicity .2-modified zirconia surface on bone tissue, the behaviour and response of MC3T3-E1 cells seeded on the surface of zirconia specimens in each group were evaluated by in vitro studies. After UV irradiation, the seeded cells were not damaged and exhibited better proliferation activity. UV-TiZ group zirconia could best promote MC3T3-E1 cell adhesion and spreading. Early cell adhesion is important for subsequent osteogenic differentiation, utilizing implant materials to recruit cells and initiate osseointegration [(To explore the effect of the UV-irradiated TiOgration [, (Li et gration .2-modified zirconia on the osteogenic differentiation of MC3T3-EI cells after UV irradiation was examined by an ALP activity test and mineralization assessment. ALP is one of the markers of early osteogenic differentiation (2-modified zirconia was further confirmed. Superhydrophilic TiO2 surfaces of biomaterials and metal implants have been demonstrated to increase bioactivity and promote osteogenic differentiation (2 surfaces can increase the recruitment, spreading, and proliferation of osteoblasts, which in turn enhances integration between bone tissue and implants (The effect of TiOntiation . The degntiation . The UV-ntiation . Notablyimplants .in vivo experiments to confirm whether the in vitro results are applicable to actual clinical applications. The in vivo results confirmed that the UV-TiZ group exhibited the best osteogenic effect. UV-functionalized dental implants are utilized clinically due to their therapeutic advantages (2 coatings appeared to be more osteoconductive after UV treatment. In further studies, we will explore the effect of UV irradiation on the formation of biofilms on TiO2-modified zirconia surfaces, which are mainly due to the presence of contaminants on material surface.Histological analysis is the criterion for evaluating implant osseointegration . Meanwhivantages . In somevantages . Althougvantages . The pos2-modified zirconia surface significantly promoted the proliferation, spreading and differentiation of seeded MC3T3-E1 cells. The in vivo evaluation demonstrated that UV-irradiated TiO2 coatings could induce more bone tissue formation around zirconia implants. Above all, the in vitro and in vivo analyses suggested that UV-irradiated TiO2-coated zirconia could improve osseointegration and will be a promising biomaterial for clinical applications.In this study, the UV-treated TiO


Machine learning analysis of factors associated with 10-year graft survival of Descemet stripping automated endothelial keratoplasty (DSAEK) and penetrating keratoplasty (PK) in Asian eyes.n = 1,335) who underwent DSAEK (946 eyes) or PK (389 eyes) for Fuchs’ endothelial dystrophy (FED) or bullous keratopathy (BK) were analyzed. Random survival forests (RSF) analysis using the highest variable importance (VIMP) factors were determined to develop the optimal Cox proportional hazards regression model. Main outcome measure was 10-year graft survival with RSF analysis of factors associated with graft failure.Prospective study of donor characteristics, clinical outcomes and complications from consecutive patients (P < 0.001). Overall 10-year survival for DSAEK was superior to PK . RSF based on VIMP (best Harrell C statistic: 0.701) with multivariable modeling revealed that BK , PK , male recipients  and poor pre-operative visual acuity  were associated with graft failure. Ten-year cumulative incidence of complications such as immune-mediated graft rejection (P < 0.001), epitheliopathy (P < 0.001), and wound dehiscence (P = 0.002) were greater in the PK compared to the DSAEK group.Mean age was 68 ± 11 years, 47.6% male, in our predominantly Chinese (76.6%) Asian cohort, with more BK compared to FED (62.2 vs. 37.8%, In our study, RSF combined with Cox regression was superior to traditional regression techniques alone in analyzing a large number of high-dimensional factors associated with 10-year corneal graft survival in Asian eyes with cornea endothelial disease. Corneal transplantation is currently the most frequently performed type of transplant worldwide , with coHowever, long-term outcomes of DSAEK compared to the traditional PK in terms of graft survival and complications such as graft rejection still vary in the published literature. Long-term studies from the Asia-Pacific , 9 and EA randomized controlled trial is not always feasible to compare DSAEK and PK, and outcomes from registry studies are valuable in providing representative results by including a large number of cases performed by several surgeons . HoweverWe collated all the data from our ongoing prospective Singapore Corneal Transplant Study (SCTS) cohort, which tracks all patients who have underwent a cornea transplant through an annual audit . Our incOur main outcome measure was graft survival, where graft failure was defined as irreversible loss of optical clarity, sufficient to compromise vision for a minimum of three consecutive months . ComplicEssentially, PK surgeries were performed using a standard technique previously described , with a P-value <0.05 was considered statistically significant. The analysis was conducted using R, version 4.0.2  with the randomForestSRC package , and operative data  . We used package , 27.P < 0.001) in our study cohort —log-rank P value < 0.001 in the remaining surviving grafts (n = 78) (P < 0.001) and FED  groups  or DSAEK (946 eyes) based on our inclusion criteria. Overall mean age was 68 ± 11 years, 47.6% male, in our predominantly Chinese (76.6%) Asian cohort with no significant differences in these baseline demographics in our PK and DSAEK groups . We had y cohort . Five-ye(n = 78) . Sub-gro) groups .P < 0.001) compared to FED and PK was a significant factor associated with graft failure more likely to fail compared to DSAEK .We ranked top variables and pair-wise interactions according to their VIMP scores  to develP = 0.011), as our study was not adequately powered to compare 10-year endothelial cell loss between groups. Complications such as graft rejection  and corneal epitheliopathy  were significantly greater in PK compared to DSAEK. There was a greater incidence of transient intraocular pressure (IOP) elevation  comparing PK and DSAEK . Complications such as wound dehiscence was unique to PK (P < 0.001) and graft detachment was unique to DSAEK (P < 0.001).Overall, we observed a greater 10-year cumulative incidence of complications in the PK compared to DSAEK group . Five-yeThe Singapore Cornea Transplant Registry prospectively collects a large database of variables and outcomes as an audit of multiple surgeons of various surgical experience, practicing with standardized surgical techniques and post-operative management . TraditiP < 0.001) and FED . These long-term graft survival results reflect the higher proportion of BK compared in FED in our Asian population, as BK was almost three times more likely to be associated with graft failure compared to FED, and BK has been shown to have poorer outcomes in both PK (P = 0.171) after adjusting for other variables. However, the RSF describes a closely associated but non-linear relationship between the donor endothelial cell count and 10-year graft survival in both PK and DSAEK  and Fuchs dystrophy (FED) in our study cohort. Similar to previous studies , 33, our both PK –36 and D both PK –39. Anotnd DSAEK .P = 0.024) in the PK group but not in the DSAEK group   . While t= 0.382) , we foun= 0.382) . Our RSFpost hoc re-analysis of the Cornea Preservation Time Study to specifically examined intra-operative complications, it was reported that surgeon and eye bank factors were the top 2 factors found to be important predictor of graft failure (P < 0.001), graft rejection (P < 0.001), and as such, less incidence of raised IOP from steroid response as the need for post-operative steroids may be reduced in DSAEK (P = 0.04).There are currently few studies that have reported 10-year outcomes of DSAEK, and to our knowledge, no reports that directly compare 10-year outcomes and complication rates of DSAEK to PK from the same study cohort. Moreover, our study used a relatively novel machine learning analysis technique to study a large number of variables while accounting for interactions and non-linear associations with a better prediction compared to traditional model development methods. Another strength is the availability of long-term graft outcomes from registry data, which can vary according to surgeon versus center experience as surgical outcomes are improved by using standardized techniques and post-operative management protocols . Compare failure . In our However, we recognize the limitations of our study which included the transition of surgical techniques from PK to DSAEK that was introduced in 2006 onward, and the reduction in number of follow-ups at 10 years. We discussed the effect of surgical experience and patient selection in our previous studies, which was mitigated by our standardized protocols and surgical techniques. Thus, we only selected primary grafts for specific corneal endothelial diseases, i.e., Fuchs dystrophy or bullous keratopathy, and previously detailed the transition of proportion of PK toward DSAEK in our study cohort . We alsoIn summary, our study provides long-term graft survival outcomes and cumulative incidence of complications, highlighting the advantages of DSAEK over PK in the treatment of end-stage corneal endothelial decompensation in Asian eyes. We used machine learning techniques to analyze the large registry database collected over a 10-year audit to determine the most important factors associated with graft failure, and used these factors to derive the optimal model for multivariate analysis, which was superior to traditional techniques. A combination of predictive (machine learning) and explanatory (regression) modeling may be a useful way of analyzing large registry datasets to examine cornea graft survival and factors associated with graft failure in future studies, which may then be used to develop a risk prediction model.marcus.ang@snec.com.sg.The data analyzed in this study is subject to the following licenses/restrictions: Anonmyized data collected as prospective audit. Requests to access these datasets should be directed to MA, The studies involving human participants were reviewed and approved by Institutional Review Board . The patients/participants provided their written informed consent to participate in this study.All authors contributed significantly to the development of the study, analysis, and manuscript preparation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Croatian Medical Journal, and Svjetlana Kalanj Bognar, the current Editor-in-Chief, have given me to write what I hope have been serious, humorous (sometimes silly), educational, and stimulating essays (“columns”) for the readership of this journal. Marko Kljaković-Gašpić, the journal’s Production Editor also has been extremely supportive and I have enjoyed exchanging views with him. Nonetheless, I have no idea how many people read what I write (“Let’s Get Something Straight”).It has been fun and I am grateful for the opportunities that Matko Marušić, a previous Editor-in-Chief of the Croatian Medical Journal in 2006 and over the next 16 years this bi-monthly  journal published 36 of them. I did not write a column for every issue because I took some time off now and again for various reasons. Sometimes it was due to my having run out of ideas or temporarily losing my sense of humor; I do not write columns as easily as summoning a taxi. First, I have an idea. Then I tell my wife what I am thinking of writing. Then she tells me what a stupid idea it is. Then I start over. If she does not tell me the proposed subject and associated opinions are stupid, I take that as approval and get to work. I have taken into account that I am writing for an audience consisting of people whose first language is not English, who may not understand American slang, whose feelings might be hurt by a sentence or phrase I meant to be humorous, or who might become angry at the Croatian Medical Journal because of something about which I misspoke. I never worried about the latter because the Editor-in-Chief and the Production Editor would point out the errors and offensiveness of my wordings, so that I could change them.I wrote my first column for the odvjetnik. Both Jelka and Franjo are now deceased but remain very much alive in my memory. We traveled together in Croatia each fall and I learned a great deal more than I taught. I remain in contact with two of Jelka’s former students, both of whom are accomplished virologists. In addition, the young daughter of a property owner on Brač where we had a study site for our tick collections, spent a year with us as a student in Colorado. My life has been better for having known these wonderful people and we were able to enter our results into the scientific record and to at least temporarily reduce the excess volumes of Dalmatian crno vino. That effort ultimately failed, but not because of any lack of trying on our parts.In reviewing my many visits to Croatia and preparing to write sophisticated columns (an effort soon abandoned), memories of individuals, places, meals, wines, history lessons, personal illnesses, geology, crops, and more came to mind. My principal scientific contact was Dr Jelka Vesenjak Hirjan, then Head of the Department of Virology of Andrija Štampar School of Public Health of Croatia, and her husband, Dr Franjo Hirjan, who was a former Justice of the Supreme Court of Croatia and was influential in our son’s decision to become a lawyer/attorney/The American comedian Woody Allen said, “I am not afraid of dying. I just do not want to be there when it happens.” I understand what he meant. I am now 86 years old and have had the usual problems of aging. I have never been 86 before, so I do not know whether upper spine surgery, lower spine surgery, cholecystectomy (followed by pneumothorax), cataract surgery, tenolysis, insertion of a cardiac stent, as well as some less dramatic insults are to be expected but they have been inconvenient. I also have developed difficulties with balance and walking and, although I have consulted with experts and tested for countless problems known to modern medicine, no one has provided me with a reason for this, other than to point out that I am 86, as though I did not know that. One physician looked at my records and declared that I am 100% healthy, except that I cannot walk. I therefore expect to be the healthiest person in the cemetery.Much worse, my older daughter died two years ago and my older sister died earlier this year, so the long-term view is becoming clearer to me. Therefore, it seems to be time to simplify what remains of my life. I have no intention of taking up golf, a game which does not involve defense, a silly lack. I have decreased the number of manuscripts I am willing to review, withdrawn my memberships in various committees, and surrendered my opportunities to criticize journals and individuals who pretend to understand virus taxonomy.A book containing 20 of my columns was published by Medicinska Naklada in 2013 and this column is sufficient to complete a second book. I find now to be a good time to quit writing and watch more baseball.hvala vam to my wife Shelley for her patience and excellent editorial assistance, to the Editors of the Croatian Medical Journal, and to whoever has read and enjoyed these columns – my gratitude to all.A hearty “Some cause happiness wherever they go; others whenever they go” – Oscar Wilde