Results: Laser-treated (Laser Mach and Laser Sint) implants exhibited the highest adhesion degree; however, limited proliferation was observed, except for Laser Sint implants, while viability differences were seen throughout the three time points, except for Ti Blast implants. Sandblasted surfaces  outpaced the laser-treated ones, inducing higher amounts of coll-I, osn, and alp, but not osc. Among the sandblasted surfaces, Ti Blast showed moderate roughness and the highest superficial texture density, favoring the most significant spontaneous differentiation relative to all the other implant surfaces. Conclusions: The results indicate that 3D cultures of stem cells on whole-body titanium dental implants is a practical and physiologically appropriate way to test the biological characteristics of the implants, revealing peculiar differences in ADSCs’ adhesion, proliferation, and activity toward osteogenic commitment in the absence of specific osteoinductive cues. In addition, the 3D method would allow researchers to test various implant surfaces more thoroughly. Integrating with preconditioned stem cells would inspire a more substantial combinatorial approach to promote a quicker recovery for patients with restorative impairments.Background: Individuals with pathologic conditions and restorative deficiencies might benefit from a combinatorial approach encompassing stem cells and dental implants; however, due to the various surface textures and coatings, the influence of titanium dental implants on cells exhibits extensive, wide variations. Three-dimensional (3D) cultures of stem cells on whole dental implants are superior in testing implant properties and were used to examine their capabilities thoroughly. Materials and methods: The surface micro-topography of five titanium dental implants manufactured by sandblasting with titanium, aluminum, corundum, or laser sintered and laser machined was compared in this study. After characterization, including particle size distribution and roughness, the adhesion, proliferation, and viability of adipose-derived stem cells (ADSCs) cultured on the whole-body implants were tested at three time points (one to seven days). Finally, the capacity of the implant to induce ADSCs’ spontaneous osteoblastic differentiation was examined at the same time points, assessing the gene expression of collagen type 1 ( Bone regeneration is limited in the elderly and individuals with pathologic disorders such as osteoporosis, diabetes, and metabolically linked diseases after trauma or surgery ,2,3,4. TThe progress in current implantology has depended on the surface functionalization of restorative materials to improve their biocompatibility and in vivo performance ,5,6. TheIn addition, in pathological settings or in the presence of elderly individuals, it is difficult to increase implant osseointegration using only physical–chemical changes because bone repair and remodeling are complicated processes involving the migration, proliferation, and differentiation of osteogenic cells . SignifiThe findings notably showed that dental implants combined with cell-based tissue engineering approaches exhibited increased cell density, extracellular matrix (ECM) content, growth factors, and stability surrounding the bone–implant contact area . PreviouOn the downside, the impact of titanium dental implants on cells varies significantly because of the different surface textures and coatings ,21,22,23As a consequence, the present study aims to examine whether the micro-geometry and surface properties of five titanium implants affect the adhesion, proliferation, and differentiation in vitro of a population of adipose-tissue-derived stem cells (ADSCs) in order to strengthen the groundwork for a more realistic combinatorial strategy and confirm the significance of testing cells in a 3D experimental condition.g for 15 min to remove the red blood cells. ADSCs were placed in Joklik modified alpha-MEM medium  supplemented with 400 U/mL collagenase type 2  equal to two times the volume of lipoaspirates and incubated for 15 min at 37 °C under moderate shaking every 2–3 min. The solution was then centrifuged for 10 min at 1800× g. After that, the supernatant was discarded, and the pellet was resuspended and filtered through a 70 μm filter. ADSCs were cultured onto 100 mm dishes (2 × 106 cells per dish) in the presence of a proliferation medium composed of α-MEM with l-glutamine 2 mmol/L, 10% fetal bovine serum (FBS), insulin 10 mg/mL, dexamethasone 10−9 mol/L, ascorbic acid 100 mM, epithelial growth factor 10 ng/mL, and gentamicin 50 ng/mL . To inhibit cell differentiation, ADSCs were kept semi-confluent, and 80% of the media was changed every three days. ADSCs were maintained at 37 °C, 5% CO2, and a humidified atmosphere in the proliferation medium. A bland detaching solution, namely CTC  in calcium, and magnesium Hank’s balanced salt solution (HBSS)), was used to detach ADSCs. Cells between passages 1 and 6 (P1 and P6) were used in the experiments [Lipoaspirates were obtained from healthy donors over the age of 18 after obtaining informed consent and following all regulatory requirements for confidentiality and the administration of biological material, according to the ethical committee of the University of Udine (Par. N. 103/2011/Sper). The donors’ lipoaspirate (15 mL) was obtained using the tumescent procedure, which involves infusing Klein’s solution and then recovering the adipose tissue. After that, the adipose tissue was centrifuged at 1800× eriments ,26.2O3, around 0.5 mm) and acid etching. According to the manufacturer, no surface debris is left from the blasting and etching procedure, which creates a uniform surface with a very high protein-binding capability before being sterilized by a plasma cold reactor and packaged under clean, low-germ conditions.Five different titanium dental implants, 1: sandblasted with aluminum oxide and acid etched ; 2: laser-sintered ; 3: sandblasted with titanium dioxide spheres and fluoride treatment ; 4: laser-machined ; 5: sandblasted with corundum and different acid etchings , have been tested and compared throughout this study. The implants had a tronco-conical shape with dimensions ranging from 3.6 to 4 mm in diameter and 8 to 9 mm in length and were chosen for their wider market representation, which could be attributed to their higher difference in roughness degree and surface texture compared to our previous research . The sur6Al4V, is created by sintering metal powder nanoparticles with one another and against the implant surface using a selective laser melting technique via a computer-controlled laser beam to produce a complex implant surface. The final texture is an intercommunicating surface that interacts with the host bone and permits osteoblastic cells to enter the implant body. The implant is dried and made ready for use after being cleaned with acetone, ethanol, and distilled water in an ultrasonic cleaner.The Laser Sint implant, made of Ti6Al4V alloy and has a moderately rough surface resulting from two subtractive, sequential manufacturing processes. The titanium dioxide sandblasting spheres (0.25–0.75 mm) give a microscale surface roughness, and the subsequent etching with hydrofluoric acid shapes the nanostructure of the implant. The implant is dried and made ready for use after being sterilized and packaged in an unspecified solution.The Ti Blast implant is made of Ti6Al4V alloy, has a sequence of finely carved, cell-sized networks manufactured through a laser subtractive process using a high-energy and highly concentrated laser beam from a CO2 laser source ranging from 10 W up to 30 W. The implant is dried, sterilized by γ irradiation, then packed and made ready for use.The Laser Mach implant, made of Ti6Al4V alloy, is etched with different acids (HCl and H2SO4) at high temperatures and then sandblasted with corundum  at high pressure (up to 5 bar) to provide a highly homogeneous surface. The implant has a high level of cleanliness, which is due to the decontamination procedure in a cold plasma reactor.The Cor Blast implant, made of extra-low interstitial TiMorphological analyses were carried out using a scanning electron microscope, Quanta250 , in high vacuum and secondary electron mode with a tension of 30 kV; the operational distance was set in order to obtain the proper magnification. Aluminum stubs covered with carbon double-sided tape were used to secure the samples. The samples were then gold sputtered with a Sputter Coater K550X .http://imagej.nih.gov, USA, accessed on 3 March 2023) [The implants were then subjected to acquisitions at higher magnifications , and three principal profiles were obtained for each implant perpendicular to the implant longitudinal axis. To evaluate the surface micro-morphology, average size, distance between the particles, and number of particles, SEM photomicrographs  were taken between two threads and, after binarization and thresholding, were analyzed using ImageJ and the nearest distances (ND) plugin (ch 2023) .Although initially created for different purposes and highlighting the measurement uncertainty and noise that can be caused by errors induced by this method and by the roughness estimation process itself ,29,30, t2 in a humidified atmosphere in the proliferation medium, were washed in PBS (Sigma), detached using CTC, and centrifuged for 5 min at 1800× g. Following this procedure, ADSCs were resuspended in the proliferation medium to a final concentration of 6.25 × 106/mL and 200 µL of medium was added at time point zero (T0) to each well. ADSCs were allowed to adhere to the implants, and after 24 h (T1), non-adherent cells were removed, transferring the implants into a clean 96-multiwell plate (Gibco) filled with fresh proliferation medium (200 µL) inside a 150mm plate. On day three (T2), the proliferation medium was changed as described for day one and left at 37 °C with 5% CO2 in a humidified atmosphere until day seven (T3) [Each dental implant (N = 9) was placed vertically in a 96-multiwell plate (Gibco), which was contained in a 150 mm plate to maintain sterility. ADSCs between passages 3 and 6, kept semi-confluent to avoid spontaneous differentiation and maintained at 37 °C with 5% CO2. Cells were then centrifuged for 5 min at 1800× g and counted in triplicate at each time point (T1–T3) in the Neubauer chamber. ADSCs’ adhesion, viability, and proliferation were evaluated in triplicate (N = 3) starting from T0 and throughout the three time points (T1–T3). Adhesion was considered as the number of adhering cells per mm2 across the time point of interest and the other time points on the respective implants. Viability was considered as the ratio of dead cells across the time point of interest and the other time points on the respective implants. Proliferation was considered as the difference in cells counted across the time point of interest and the other time points on the respective implants. The total number of cells seeded at T0, as well as the surface of the submerged implant, were used to normalize the adhesion, viability, and proliferation assessment results [The procedure for cell detachment from the implants involved transferring the implants into 1.5 mL conical tubes containing CTC, followed by 10 min of incubation at 37 °C in a humid environment with 5% CO results ,26.g, total RNA was extracted from ADSCs cultured on each different implant (N = 3) using TRIzol (Gibco). RNA samples were then quantified by spectrophotometer and treated with DNase I . First strand cDNA synthesis was performed with 100 ng of total RNA using M-MLV reverse transcriptase (Invitrogen). PCR amplification was performed in a final volume of 50 µL, using 10 ng of cDNA, 50 pmol of each primer, and 2 U/µL of TaqI polymerase . The following primers were employed to quantify the expression of osteoblastic markers on days one, three, and seven in triplicate: alkaline phosphatase  GCAGGCAGGCAGCTTCAC, TCAGAACAGGACGCTCAGG, 496 bp, 60.5 °C, NM_000478.3; collagen Type I (coll-I) TAAAGGGTCACCGTGGCT, CGAACCACATTGGCATCA, 355 bp, 60 °C, NM_000088.3; osteocalcin (osc) TCACACTCCTCGCCCTATTG, CTAGACCGGGCCGTAGAAG, 293 bp, 58 °C, NM_199173.3; and osteonectin (osn) CACAAGCTCCACCTGGACTA, GAATCCGGTACTGTGGAAGG, 525 bp, 58 °C, NM_003118.2. RNA polymerase type II GCACCACGTACACCAATG, GTGGGGCTGCTTTAACCA 350 bp 56 °C NM_000937 was used as a housekeeping gene  [After being washed in PBS (Sigma), detached using CTC, and centrifuged for 5 min at 1800× erg, DE) ,26.t-test was used to compare data from similar groups. Statistical significance was determined by utilizing SPSS 23.0  to analyze the data, and significance is indicated as + Al Blast, * Laser Sint, £ Ti Blast, % Laser Mach, and ^ Al Blast (ANOVA followed by Bonferroni’s post hoc test), and * T1 and 24 h–3 days, ^ T2 and 24 h–7 days, and % T3 and 3–7days (paired t-test) with p ≤ 0.05 or p ≤ 0.001.The data are presented as mean ± standard deviations (SD) for cell counts and gene expression, implant roughness, and surface particle characteristics. The statistical significance was ascertained using the analysis of variance (ANOVA) and Bonferroni’s post hoc tests. A paired ADSCs were isolated from lipoaspirates taken from healthy donors using the tumescent technique as described in our previously published works ,26.In summary, flow cytometry was used to show the stemness-related membrane proteins in ADSCs at passage three (P3), demonstrating the presence of CD73, CD90, and CD105, but not the hematopoietic CD34, CD45, and CD106, as previously reported . To valiSEM microphotographs of the implant surfaces revealed their characteristic morphology . The texThe microarchitecture of a biomaterial, including the number of particles and their size, distribution, and density, dramatically affects its interaction with tissues and cells and influences the mechanical properties of the scaffold itself . In partp > 0.05). The Ti Blast implant displayed a slightly lower mean distance between particles (4.06 ± 0.3 μm) than the Laser Mach (5.4 ± 0.76 μm) and Cor Blast (6.17 ± 1.17 μm) implants (p ≤ 0.05) (The results demonstrated no significant difference in the mean area of the surface particles between the tested implants  (p > 0.0 ≤ 0.05) .p ≤ 0.05) (The nearest particle distance of the Al Blast (4.26 ± 0.38 μm), Laser Mach (4.34 ± 0.49 μm), Cor Blast (4.86 ± 0.69 μm) and Laser Sint (4.07 ± 0.2 μm) implants was similar, while in the Ti Blast implant (3.5 ± 0.22 μm), the distance was significantly smaller than the rest ( ≤ 0.05) .p ≤ 0.05) (2) implants (p ≤ 0.05). The number of surface particles on the Al Blast (250 ± 1.5), Laser Sint (304 ± 39), and Cor Blast (174 ± 69) implants were different from each other (p ≤ 0.05) but no significant differences were seen with respect to the Laser Mach implant (273 ± 126)  was the smallest and was significantly lower than in the Cor Blast (2.93 ± 1.21 μm) and Laser Mach (2.24 ± 0.74 μm) implants, but the difference was not significant with respect to the Al Blast (2.12 ± 0.76 μm) or Laser Sint (1.98 ± 0.3 μm) implants ( ≤ 0.05) . The res3 ± 126) .Surface roughness of titanium implants, particularly moderate roughness, allows for improved cell and bone connections ,32,33 anp ≤ 0.05 indicating significance (ANOVA followed by Bonferroni’s post hoc test) , root mean square deviation (Rq), skewness of the assessed profiles (Rsk), and kurtosis of the assessed profiles (Rku) , substanoc test) .p ≤ 0.05) (p ≤ 0.05)  and Rq (2.04 ± 0.04 μm and 2.04 ± 0.1 μm), respectively  . In cont ≤ 0.05) .p ≤ 0.05) (p ≤ 0.05)  , whereas ≤ 0.05) .The surface texture and roughness analyses revealed that the Ti Blast implant had the highest particle density , even though there were no appreciable differences in their area. Instead, as seen by the corresponding indicators (Ra and Rq), the Al Blast and Cor Blast implants exhibit the greatest degree of roughness, whereas the Laser Mach implant had the lowest values. As expected, the Laser Mach implant also displayed the highest Rsk and Rku values, as opposed to the Al Blast implant.6 cells) and the surface area of the implants (p ≤ 0.001 indicating significance (ANOVA followed by Bonferroni’s post hoc test and paired t-test).In order to address any issues related to the seeding strategy, the ratios of adherent cells retrieved at the three time periods were standardized according to the total number of cells at day zero (1.25 × 10implants A–D. The 2), followed by the Laser Sint (152.8 ± 36.4 cells/mm2), Ti Blast (87.1 ± 20.1 cells/mm2), Al Blast (65.3 ± 30 cells/mm2), and lastly, Cor Blast (59.2 ± 48 cells/mm2) implants (p ≤ 0.001). The Cor blast, Al Blast, and Ti Blast implants exhibited the lowest adhesion with respect to the remaining surfaces (p ≤ 0.001)  .2) (p ≤ 0.001) (2) (p ≤ 0.001) or remained stable on the other implants, including the Laser Sint (127.4 ± 62.1 cells/mm2), Al Blast (67.3 ± 37.6 cells/mm2), and Ti Blast (79.2 ± 56.2 cells/mm2) implants  , whereasimplants .p = 0.001) and Al Blast (p ≤ 0.001) implants; similarly, the Laser Sint implant differed from the Ti Blast (p = 0.032) and Al Blast (p = 0.002) implants. The Ti Blast and Al Blast implants differed from the Laser Sint and Cor Blast implants   .2) (p = 0.008) implants, whereas on Laser Mach (138.2 ± 83 cells/mm2) implants, the increase was not significant. On Al Blast (35.7 ± 16.2 cells/mm2) implants, the cell number exhibited a significant reduction (p ≤ 0.001), while the reduction was not significant on Cor Blast (114 ± 64.2 cells/mm2) and Ti Blast (62.7 ± 40.6 cells/mm2) implants , Laser Sint (p ≤ 0.001), and Cor Blast (p = 0.024) implants (p ≤ 0.001). The Laser Mach implant showed adhesion differences with the Ti Blast (p ≤ 0.001), Al Blast (p ≤ 0.001) and Laser Sint (p = 0.003) implants, but not with the Cor Blast implant , and from the Laser Mach implant (p = 0.003). The adhesion on the Cor Blast implant was considerably different from those on the Al Blast, Laser Sint (both p ≤ 0.001), and Ti Blast (p = 0.024) implants , Al Blast (p ≤ 0.001), and Cor Blast (p = 0.006) implants, and an increase on the Laser Sint implant (p = 0.008)  .p ≤ 0.05) (p ≤ 0.001), but not in the Laser Mach implant (0.29 ± 0.94) . Lastly, at day seven, the viability of the ADSCs cultured on the Laser Sint (0.32 ± 0.54) and Cor Blast (0.57 ± 0.92) implants was significantly different from those on the Al Blast (−0.88 ± 0.83) and Ti Blast (−0.36 ± 0.64) implants , but not from those on the Laser Mach implant (−0.16 ± 0.55) (p > 0.05) (The cell viability at the 24 h time point with respect to the different implant types demonstrated that the Cor Blast implant (1.7 ± 1.6) revealed better viability with respect to the Al Blast (0.03 ± 0.56), Laser Mach (−0.34 ± 0.37), Laser Sint (−0.22 ± 0.62) and Ti Blast (−0.085 ± 0.69) implants  . The res ± 0.94) ; meanwhi > 0.05) .p = 0.016, p = 0.026, and p ≤ 0.001), while this decrease was noticeable on the Cor Blast implant only at three days (p ≤ 0.001) and on the Laser Mach implant only at 24 h (p ≤ 0.019)   . The celven days . Finally > 0.05) .In summary, a distinctive difference in adhesion was highlighted for laser treated implants (Laser Mach and Laser Sint), which exhibited the highest degrees; however, limited proliferation was observed, specifically for the Laser Sint implant, and viability differences, except for with the Ti Blast implant, were seen throughout the three time points.p ≤ 0.05 indicating significance (ANOVA followed by Bonferroni’s post hoc test). By enhancing the expression of osn, coll-I, and alp in comparison to control cells, all of the treatments and surfaces of the whole-body implants were able to promote osteoblastic differentiation, as shown by the results. The Al Blast (152.8 ± 5.6 fold change (FC)), Ti Blast (120.1 ± 4.9 FC), Cor Blast (106.5 ± 7 FC), Laser Mach (66.2 ± 0.73 FC), and Laser Sint (62.9 ± 2.9 FC) implants triggered the highest coll-I expressions when compared to the control at the 24 h time point (p ≤ 0.05)  .osn expression at 24 h reached the highest value on the Laser Mach implant (14.4 ± 0.4 FC), followed by the Cor Blast (12 ± 0.3 FC) and Laser Sint (7.4 ± 0.3 FC) implants (p ≤ 0.05), while on the Ti Blast and Al Blast implants it was not detected  (p ≤ 0.05) and osn  expressions when compared to the other implant types at the same time point (p ≤ 0.05)  .alp messenger  compared to the Laser Sint (24.9 ± 0.25 FC), Laser Mach (11.4 ± 0.3 FC), and Cor Blast (22.9 ± 1.5 FC) implants (p ≤ 0.05)  .coll-I  (p ≤ 0.05) and osn  (p ≤ 0.05) (p ≤ 0.05)   compared ≤ 0.05) .alp gene was not induced by the Al Blast implant, as at 24 h, but it reached the highest expression peak under the stimulation of the Ti Blast (78.4 ± 2.6 FC) surface, which was significant compared to the Laser Sint (22.1 ± 0.8 FC), Laser Mach (19.3 ± 0.5 FC), and Cor Blast (41.3 ± 0.7 FC) implants (p ≤ 0.05) (The  ≤ 0.05) .osc could not be induced by any surface.It is interesting to note that by day seven, the late osteoblastic differentiation marker alp synthesis at 24 h and three days, it was able to induce its expression at seven days  . The Cor Blast implant showed significantly increasing alp, osn, and coll-I at the 24 h–three day and three–seven day intervals, but was not able to induce a substantial increase in alp and coll-I during the 24 h–three day interval   . In continterval . On the  ≤ 0.05) .p ≤ 0.05) , but was able to be inductive for alp only during the three–seven day interval (p ≤ 0.05) and coll-I at 24 h–three days (p ≤ 0.05) and three–seven days (p ≤ 0.05)  . The Las ≤ 0.05) .coll-I, osn, and alp messengers, although not osc. Significantly, among the sandblasted surfaces, the Ti Blast surface promoted, in general, the strongest expression of coll-I, osn, and alp.Overall, the results showed that sandblasted surfaces  outperformed the laser-treated ones, promoting the highest spontaneous osteoblastic induction and synthesizing the highest levels of Since titanium dental implants have a wide range of surface textures and coatings, their impact on cells varies significantly. However, since 3D cell cultures on whole dental implants more closely resemble and mimic physiological cell interactions, they have been proven advantageous in assessing implant capabilities ,25. As aWe found that while roughness is still significant, other factors sensibly influence the adhesive potential of the surfaces of whole-body titanium dental implants. Specifically, the range of surface roughness between Ra 0.8 and 1.2 µm and Rq 1.4 and 1.8 µm does have a beneficial impact on the adhesion of ADSCs.Implant surfaces within this range of roughness and surface particle texture, such as those found on the Laser Sint and Laser Mach implants, favor a reasonable degree of adhesion compared to the other tested implants, similar to what we previously demonstrated . We alsoSeveral studies have demonstrated that the surface characteristics of the dental implants are impacted by manufacturing procedures, changing their composition and affecting the function of tissue and cells ,36. IndeAccording to these findings, the implants that had undergone laser-related treatments, such as the Laser Sint implant, and to a lesser extent the Laser Mach implant, as well as a laser-treated implant from a previous study , appearecoll-I and osn. However, in general, sandblasted implant surfaces, such as the Ti Blast, Al Blast, and Cor Blast surfaces, were more osteoinductive than laser-treated ones. Interestingly, the sandblasted surfaces, Al Blast, Cor Blast, and Ti Blast, underwent treatments involving aluminum, corundum, and fluoride, respectively. Aluminum oxide and corundum  are deposited during the sandblasting process and seem to have differential effects according to their concentrations. In fact, it was pointed out that high concentrations of residual Al reduce mineralization and osseointegration, while lower concentrations [The differences in the surfaces and textures showed an influence on ADSCs’ spontaneous differentiation; in this case, the Ti Blast implant, but secondarily the Al Blast implant with some deficiencies, demonstrated the highest inductive potential on the osteoblastic markers trations ,44 have trations  and may trations  and stemtrations ,46 and itrations ,47,48,49The findings also showed that the Ti Blast Implant, created by sandblasting with titanium dioxide spheres and etching with hydrofluoric acid, showed no significant effects on adhesion and viability despite its moderate roughness. However, among the other implants, it presented the highest density of surface particles, also suggesting the importance of the texture density.As an explanation, the presence of this high-density texture may not have been as appealing in terms of cell adhesion, but along with the presence of fluorine ions derived from the surface treatment, it may have been very important in inducing ADSCs’ spontaneous differentiation  via the 10(PO4)6(OH)2) coating’s compositional and structural similarity to bone [Previous effects become less consistent when considering that, from our previous results, the implant manufactured through hydroxyapatite and bland acid etching (HA Blasted) performed considerably better than all other implants despite having a similar texture and roughness . Therefo to bone  enhances to bone ,57, stim to bone  via inte to bone ,58. On t to bone ,60.The adhesion and proliferation of the laser-treated implants exhibited the highest degree, possibly due to the influence of the post-processing methods.The sandblasted implant surfaces performed better in terms of osteoinduction, possibly via the chemical treatments the implants underwent during the manufacturing process.The Ti Blast implant’s osteoinductive capability might have benefited from the highest texture density and the fluorine surface treatment.This study’s results align with previous findings, demonstrating that stem cells are actively influenced by the topography and surface treatment of whole-body implants and the 3D culture approach is capable of unveiling the potential of both ,25. IndeIn light of these results, it is therefore recommended to pursue further research using the 3D culture methodology to broaden and better understand the effects and differences of implant surfaces’ antimicrobial activity, wettability, coatings, or treatments with biologically active molecules, as well as various materials such as zirconia, tantalum, or porcelain.Finally, by implementing this 3D approach and the subsequent advancement of the combinatorial approach in conjunction with pre-clinical contexts, it would be possible to identify the most efficient and appropriate surface to be used in combination with preconditioned stem cells, promoting a more significant and faster recovery while significantly improving the healing process in individuals with restorative deficiencies.


Saccharum officinarum L.exploitationand processing result in different byproducts, such as filter cake(FC). This study aimed to establish the most suitable experimentalconditions to obtain lipophilic bioactive compounds from FC industrialresidues, considering their high efficiency, cost-effectiveness, extractionyield, composition, and physicochemical properties. Results indicatedthat the most appropriate methodology consisted of the pretreatmentof the FC sample with H2SO4, followed by ethanolicextraction (B6 method), avoiding energy-consumption FC drying stepsand providing ethanol recovery (approx. 90%). The obtained B6 extractyield was 9.59 ± 0.27 g/100 g of FC dry weight, and this methodologyproved to be more efficient in obtaining fatty alcohols (20.28 ±1.48 g/kg extract) and phytosterols (31.56 ± 0.18 g/kg extract)while maintaining lower total monosaccharide concentration (26.19± 1.82 mg/g extract). Furthermore, the geographically relatedmultivariate analysis in wax composition and antioxidant activitywas evaluated by comparing B6 waxes from Guariba (G) and Univalem(U), both provided by Brazil and collected in June 2020. Overall,the wax composition is affected, but the antioxidant activity is uncompromised,which indicates that the optimized wax extraction method can be appliedto FC. Ethanolic extraction of thefilter cake industrial byproductsupports the implementation of circular economy methodologies appliedto the research and development of sustainable lipophilic materials. Saccharum officinarum L. (sugarcane).It is a slurry that emerges from sugar and ethanol production industriesand is currently used as fertilizer in sugarcane fields due to itsabundance in minerals and organic matter.2 Furthermore,as sugar is produced by extracting the juice from crushed sugarcane,several byproducts besides filter cake including sugarcane straw,bagasse, and vinasse are released during this process.3Filter cake (FC) is a byproduct that resultsfrom the processingof 4 Brazil and other countries such as Pakistan are increasingsugarcane production, making the filter cake industrial output 2.7million tons during 2022/2023.5 An important environmentaland economic concern is how to dispose of filter cake, since it representsan approximate production of 30 kg/ton of milled sugarcane.6 Currently, most sugarcane waste biomass is burned, unused, or discarded, and it is crucial to adoptinnovative circular economy policies in the sugarcane industrial sectoras a key strategy to reduce, reuse, and recycle these generated biowastes.7With a global sugarcane production of 72.91 tons/ha, the topproducerscontribute to approximately 1.75 billion tons annually, Brazil beingthe main leader in the manufacture of ethanol sector.8Unprocessed filter cake contains approximately 75% of water,andits composition depends on several factors: type of soil, sugarcanevariety, harvesting method, juice extraction, and other products usedfor clarification and filtration methods. Furthermore, the filtercake can be used to produce bio-wax as an alternative to other vegetable,animal, and synthetic waxes. Such materials can be applied to pharmaceutical,chemical , orcosmetic  industrial fields.9 Thus, phytosterols are valued as antioxidants due to their freeradical scavenger capacity.10 Moreover,the hypocholesterolemic effects of phytosterols and the anti-obesityproperties of octacosanol have also been evidenced in previous studies.12 In addition, other less-apolar compounds such as phenolic acids,anthocyanins, and carotenoids can be present in vegetable plants.Usually, they are involved in plant adaptation to adverse environmentalconditions and are considered antioxidant agents.13Previous studies identified long-chain aliphatic primary alcohols(policosanol), phytosterols , triterpenoids,wax esters, aldehydes, and fatty acids in sugarcane-derived waxes.14 Greener/sustainable procedures that bypass the handling of thesesolvents should be preferentially chosen.15 Bio-based ethanol from sugarcane biomass feedstock is classifiedas a biodegradable and low-toxicity solvent, and it has proven itscost-effectiveness and high efficiency in extracting target bioactivecompounds.16 Boiling condensation extractioncycles can improve extraction yields and lead to fewer solvent requirements.15 The assessment of extraction parameters suchas time and temperature is crucial, as they greatly influence theeffective yields of the bioactive molecules in extracts.17Previous efforts in the chemical and engineeringsector are beingmade in order to prevent the utilization of petroleum solvents suchas hexane, dichloromethane (DCM), benzene, and toluene, which arehighly volatile extractants that contribute to climate change, pollution,and hazard manufacturing health problems.19 Generally, epicuticular wax compounds increase in plants in response to abiotic stress .20During the first decade of the XXI century,the high requirementfor sugar and ethanol production led to the expansion of sugarcanecrops in Brazil. The concentration of sucrose, resistance to pests,and water stress are some varying factors of the local climate, soiltype, and chemical, physical, and morphological attributes.S. officinarum L. crops in Brazil that are approximately 300 km apart, namely,Guariba and Univalem, both collected in June 2020.This studyaimed to evaluate the optimal filter cake wax isolationconditions, using boiling methods to contribute to the valorizationof this waste stream. Seven different ethanolic methods were usedto extract wax from the filter cake byproduct attending to differentpretreatment, extraction temperature, and solvent mixtures. Afterthe selection of the better-suited extraction method, the geographicallyrelated variation in extracts was assessed within two different batchesof raw filter cake provided from different S.officinarum L. crops in Brazil .For the lipid extraction optimization, the Guariba batch collectedin November 2019 was used, and for the study of geographical variation,Guariba and Univalem batches both collected in June 2020 were used.Sugarcane filter cake was providedby Raízen  from different 2SO4, 95.0–98.0%) purchased from Merck .Ethanol  was purchased from PanReacAppliChem . For boiling pretreatment, tap waterwas used and, when required, acidified with sulfuric acid (HFor high-performance liquid chromatography (HPLC)analysis, 2-propanol(LC–MS Grade ≥99.9%), isooctane (HPLC Grade ≥99.8%),and dichloromethane (DCM) (HPLC Grade ≥99.9%) were purchasedfrom VWR Chemicals ; acetone (HPLC Grade ≥99.8%)from Thermo Fisher Scientific , ethyl acetate(HPLC Grade ≥99.7%) and water from Honeywell , acetic acid (HPLC Grade≥99.8%) from Carlo Erba Reagents , and triethylamine(TEA) (≥99.5%) from Merck .N,O-bis(trimethylsilyl)trifluoroacetamidewith 1% trimethylchlorosilane (BSTFA) were purchased from Merck .For gaschromatography–mass spectrometry (GC–MS)analysis, the analytical standard tetracosane (99%) and the derivatizingreagent 4), ammonia solution , 1-methylimidazole (99%), acetic anhydride (99%), anhydrousacetone (99.9%), and 2-deoxy-d-glucose (≥98%), obtainedfrom Merck .For neutral monosaccharide quantification, sampleswere treatedwith sodium borohydride (NaBH2PO4) anhydrous, ≥98%,purchased from Merck  and Trolox  obtained from Merck .During the antioxidant experiments,phosphate buffer solution  was prepared by using the salt solution of sodium dihydrogenphosphate , from Thermo Fisher Scientific , ABTS (≥98%),potassium persulfate ,and 2,2′-azo-bis-(2-methylpropionamidine)-dihydrochloride  were purchased from Merck  as well as thefluorescein disodium salt. For total phenolic content determination,Folin–Ciocalteu’s phenol reagent (FCr) and sodium carbonate were obtained from Merck .DPPHThe human keratinocyte cell line HaCaT was purchasedfrom CLS—CellLine Services (no 300493), and Dulbecco’s modified Eagle mediumfrom Thermo Fischer  supplemented with10% fetal bovine serum (FBS)  and 1% penicillin–streptomycin antibiotic . For cytotoxicity, the PrestoBlue assay was used. For cell culture,dimethyl sulfoxide  was used and purchasedfrom Merck .2SO4 (aq.) 0.33% w/v solution boiling; extractiontemperature , and sample/solvent proportion(1:10 or 1:20 w/v). Further, depending on the extraction method, filtercake samples were dried at 55 °C on a Thermo Fisher ScientificOven  until constant weight (15 h). A summaryof the assayed isolation methods is depicted in Filter cake samples were subjectedto different ethanolic lipid extraction conditions, Soxtec (S) andBoiling (B), varying sample pretreatment conditions: i.e., water boilingor HIn the procedure labeled as S method and used as areference, thelipophilic extraction was carried out in triplicate using a Foss SoxtecTM8000 apparatus . Within this method, previouslydried FC samples were extracted with EtOH at the proportion of 1:20(w/v) at 130 °C for 2 h of boiling followed by 1 h of rinsingat atmospheric pressure. Afterward, the resultant extraction solutionwas evaporated in a rotary evaporator Heidolph HeiVAP  under reduced pressure  ina temperature-controlled bath at 40 °C.2SO4 (aq.) 0.33% w/v solution.The boiling methodsidentified as B1–B5 were performed usingpreviously dried FC, while the B6 method was performed using wet FC(about 68.19% humidity). Moreover, boiling methods B1–B3 wereperformed without FC pretreatment, while B4 included a pretreatmentprocedure with water, and B5 and B6 involved a pretreatment with aHIn general,these methods consisted of the mixing of FC and solventat different proportions in a 500 mL round bottom flask that was keptunder reflux in a silicone oil bath at different temperatures withconstant magnetic stirring for different time periods [3 h (withoutpretreatment) or 6 h (including FC pretreatment)]. Afterward, themixture was filtered while still hot through a synthetic fabric filter(Tescoma) using a porcelain Büchner funnel under vacuum andthen evaporated (as described for the Soxtec method).2SO4 (aq.) 0.33% w/v solution (B5 and B6) at a sample/solvent proportionof 1:20 (w/v), placed in a silicone oil bath at 120 °C, underreflux and constant magnetic stirring for 3 h. After that, the mixturewas filtered as described above, and the obtained filter cake driedat 55 °C for 15 h (except for method B6 in which FC was not driedafter the pretreatment). Finally, the pretreated FC was extractedwith ethanol under the same conditions as B1.Extractionmethods B1 and B2 were performed using the same extractionsolvent, EtOH, at the same temperature (85 °C), only varyingthe sample/solvent (w/v) proportion, which was 1:20 (w/v) for B1 and1:10 (w/v) for B2. Within the B3 method, a mixture of ethanol/waterat 1:1 (v/v) was used as extraction solvent; the sample/solvent ratiowas 1:20 (w/v) and the temperature was set at 105 °C. Regardingboiling methods B4–B6, all included an FC pretreatment thatconsisted of previous aqueous extraction of the FC samples beforetheir ethanolic extraction. In a 500 mL round bottom flask, the FCsamples were mixed with water (B4) or a HIn the end, allof the obtained extracts (S and B1–B6 methods)were completely dried, until constant weight, in a vacuum oven at60 °C and 100 mbar and then weighed, and the respective yieldswere assessed.l-rhamnose, fucose, d-ribose, arabinose, xylose, mannose, galactose, and glucose)was performed by polysaccharide reduction and acetylation in alditolacetates according to Pinto et al.21 andFaustino et al.22 Therefore, 2–3mg of the sample was accurately weighed into glass tubes, and polysaccharideswere hydrolyzed with 2 M H2SO4 before the derivatizationreaction. Monosaccharides were reduced with NaBH4 (15%in NH3 3 M) and then acetylated with acetic anhydride inthe presence of 1-methylimidazole. The mixture was centrifuged forphase separation, the aqueous phase was removed, and analytes wereseparated and detected by GC-FID using a DB-225  capillary column . For the quantificationof neutral monosaccharides, 2-deoxyglucose (200 μL at 2 mg/mL)was used as an internal standard.The quantification ofneutral monosaccharides  mode at 70 eV, a source temperature of280 °C, a transfer line at 300 °C, and a quadrupole in ascan range from 33 to 1000 amu per second. The identification wasbased on the comparison of the obtained mass spectra with the informationon the NIST Library (v.2.3).For the analysis by GC–MS, samples were derivatized intotheir trimethylsilyl (TMS) derivatives. In a glass vial, samples wereaccurately weighed (5 mg), and 100 μL of tetracosane/internalstandard (0.5 mg/mL in DCM), 30 μL of BSTFA, and DCM to a finalvolume of 1.3 mL was added. The mixture was incubated for 60 min at30 °C. The derivatized samples were analyzed on a GC–MSmodel EVOQ  coupled to a mass spectrometer,with a Rxi-5Sil MS column (30 m × 250 μm × 0.25 μm)at a constant flow of 1 mL/min. The carrier gas used was helium, andthe GC–MS conditions were as described by Teixeira et al.23 with some modificationsdescribed by Teixeira et al.18 The flowrate was set at 0.275 mL/min with an injection volume of 20 μLand the detector evaporator and nebulizer temperature was set at 60°C with nitrogen as the nebulizing gas at a 1.20 SLM flow rate.In all performed analyses, all of the samples were injected at leastin triplicate.The samples were prepared to a concentrationof 3 mg/mL in DCM and afterward analyzed on an HPLC  attached to an EvaporativeLight Scattering Detector  using nitrogen as the nebulizing gas coupled to aZorbax RX-SIL column . Analysis conditions were assayed as described byAbreu et al.,–1, with a resolution of 4cm–1, by accumulating 16 scans.24 The FTIR- ATR vibrational bands were identified based onthe literature.25The samples were analyzed on a PerkinElmerParagon 1000 FTIR  with the ATR accessory(Diamond/ZnSe). The spectra were obtained in the wavenumber rangeof 4000–550 cm2 flow of 40 mL/min,as already described by Teixeira et al.24 First, the samples were heated from 20 to 130 °C to eliminatethe sample’s thermal history.26 Then,a cooling step to −10 °C and a second heating step, from−10 to 500 °C, were performed at a constant rate of 10°C/min. Only the transitions observed during the cooling andthe second heating cycles were considered.The thermalcharacteristics of the samples  were measured on a 204 F1 PhoenixDSC . The samples were weighed (4 mg) into a pierced-lidaluminum pan and analyzed under an N•) microplate method, the free radicalscavenging activity was determined spectrophotometrically and performedin a Greiner Bio-One transparent 96-well microplate (North Carolina).The method was assayed as described by Bobo-García et al.27 with some modifications described by Teixeiraet al.24 Briefly, samples were preparedin different concentrations, namely, from 7.0 to 0.20 mg/mL in methanol.Absorbance variation at 515 nm after the addition of samples or standardwas measured in a Synergy H1TM microplate reader . The inhibition capacity is expressed as IC50 values. IC50 (mg/mL)was calculated by For the 2,2′-diphenyl-1-picrylhydrazylradical  was calculated using eq The 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonate)(ABTS) assay was performed in a Greiner Bio-One transparent 96-wellmicroplate (North Carolina), based on the inhibition by antioxidantsof the absorbance of the radical cation ABTS31 with some modifications evidenced by Teixeiraet al.24 The fluorescence values were recordedevery minute over the incubation period at 458 and 520 nm using aSynergy H1TM microplate reader , and theexperiment was carried out in triplicate.To perform the oxygen radical absorbancecapacity (ORAC) microplate method, samples were prepared from a stocksolution (2.5 mg/mL) with several subsequent sequential dilutionsfrom 2.00 to 0.06 mg/mL in phosphate-buffered saline . The stock solution was submitted to ultrasound (10 min) toimprove the dissolution and then filtered using Macherey–Nagel0.45 μm pore size Chromafil PET filters .The procedure was assayed as described by Dávalos et al.32 where the ORACvalue (μmoles TE/L) corresponds to the x valueobtained from Trolox linear regression (y = mx + b) by replacing y from AUC sample values. DF: dilution factor. L solvent/g sample:volume of the prepared mother solution/mass of the sample mother solution.The antioxidant capacitywas expressed as ORAC value in μmoles of Trolox equivalents(TE) per gram of the sample, and 33 a modified Folin–Ciocalteu’s(FCr) method.34 Samples were prepared inethanol at a stock concentration of 20 mg/mL and then diluted andmixed (50 μL) with 2.5 mL of diluted FCr 1:10 (v/v) and 2 mLof a hot Na2CO3 saturated solution. After incubationfor 5 min at room temperature in the dark, the absorbance was determinedat 760 nm in a UV-1900 UV–vis spectrophotometer . The total phenolic content was expressed as mg GAE/gof the sample  by comparison with a calibrationcurve with gallic acid standards from 25 to 800 μg/mL.The determination of thetotal phenolic content assay was performed as described by Papottiet al.,2 in a humidifiedatmosphere. The cytotoxicity of lipidic extracts on human immortalkeratinocytes (HaCaT) was evaluated using a PrestoBlue  assay according to the manufacturer’sinstructions. Cells were seeded at 1 × 104 cells/wellin 96-well plates and exposed to lipidic extracts at different concentrations(2.50–0.16 mg/mL) diluted in Dulbecco’s modified Eaglemedium  for 24 h, inquadruplicates. Wells with media supplemented with lipidic extracts(without cells) were used to subtract a possible influence of thesamples in the PrestoBlue fluorescence signal. Cells treated with10% DMSO  wereused as a negative control. Afterward, the PrestoBlue reagent wasadded to the media and incubated for 2 h. The fluorescence signalwas read in a Synergy H1 microplate reader .Results are expressed in the percentage of metabolic inhibition incomparison to the control (cells without treatment). At least twoindependent experiments were performed.The humankeratinocyte cell line HaCaT (CLS—Cell Line Services—300493)was kept in culture in Dulbecco’s modified Eagle medium  supplemented with 10% fetalbovine serum (FBS)  and1% penicillin–streptomycin antibiotic  at 37 °C, with 5% COResults are reported as mean values ±standard deviation (SD). Data were first analyzed for normality distribution. Levene’s test was applied to verifythe homogeneity of the variances. Afterward, a one-way analysis ofvariance (ANOVA) test was applied with the Tukey post hoc test todetermine differences within groups. For a two-group comparison, aStudent’s t-test was assayed. The level of significance wasset in general at 0.05. Analyses were performed with the aid of IBMSPSS Statistics software .https://www.metaboanalyst.ca/).For principalcomponent analysis (PCA) and Heatmaps, the web-based tool suite Metaboanalyst(v5.0) was used , and selective lipid composition.The obtained results regarding isolation yields after using differentextraction conditions on filter cake samples are shown in These differences in the isolation yield highlightthe importanceof both solvent proportion and temperature. Thus, while method S wascarried out at 130 °C with a sample-to-solvent ratio of 1:20,procedures B1 and B2 were performed at 85 °C with ratios of 1:20(B1) and 1:10 (B2). These latter results suggest that given the sametemperature (85 °C for B1 and B2), the decrease of ethanol inthe extraction mix harms the extract yield. Thus, for the same sample-to-solventratio, higher temperatures will increase extraction efficiency . However, in order to understand what differences are associatedwith variations in yield, it is essential to study the compositionof all extracts.Since filter cake is a clarification byproductof sugarcane juice,some monosaccharides may be retained and eventually recovered by theassayed processes of this work. Thus, further tests with mixturesof ethanol/water as well as aqueous washing of filter cake prior tothe extraction attempted to increase the purity of the lipid extractsby decreasing sugar content.35Saccharides such as rhamnose, arabinose,xylose, mannose, glucose,and galactose have already been identified in the water-soluble fractionof fruit waxes.Interestingly, method B3, which used an ethanol–watermixture(1:1) in a 1:20 sample/solvent ratio, was the only procedure ableto recover galactose, glucose, mannose, and arabinose. This suggeststhat saccharides must be previously eliminated before proceeding withlipid isolation to increase the lipidic extract purity. Thus, to recoverlipids from filter cake using ethanol, a previous extraction stepwas studied using water to eliminate monosaccharides.p ≥ 0.05). Also, theB4 method resulted in significantly lower total sugar content (47.08± 6.37 mg/g extract), p ≥ 0.05) to those of B2 . These latter results may suggestthat although ethanol can dissolve sugars, the amount of solvent usedin the extraction is also a key factor in defining extract composition.The lowest total monosaccharide content was obtained when assayingmethods B5 and B6 (such procedures are in the group of the ones usingwater prewash), and extraction yields were 7.55 ± 0.85 g extract/100g and 9.59 ± 0.27 g extract/100 g, respectively , resulting in higher standarddeviation in some of the assays analyzed for these extracts.Thus, the reported data regarding extract yield and sugar compositionfor each wax highlights the impact of the assayed procedure. Hence,in order to select the most suitable isolation conditions, more informationabout the effect of each method on the lipid profile composition iscrucial.In the assayed chromatographic conditions, it was possible to identifydifferent compounds such as fatty acids, fatty alcohols, phenolicacids, aldehydes, and phytosterols.After evaluating the lipidcomposition of the extracts obtained with the different extractionmethods by GC–MS 4, fatty Regarding the rest of the samples, as listed in 37 1-octacosanol ,38 and β-sitosterol (phytosterols).39According to the results, the lipid extractscontained interestingbioactive compounds previously described by other authors: coumaricacid (phenolic acid),40 In fact, coumaric acid was present in B5 extractsat 2.04 ± 0.24 g/kg and in B6 at 1.60 ± 0.08 g/kg.It has been described that the phytochemistry of sugarcanewaxincludes phenolic acids that are considered antioxidant agents.41 Furthermore, the S and B6 samples had the highestconcentration of phytosterols, 32.77 ± 1.39 and 31.56 ±0.18 g/kg, respectively.Previous studies also reported that sitosterol is the main sterolin plants, but stigmasterol and campesterol also occur in nature.42 In the current study, 1-octacosanolwas present in all extracts, varying from 10.35 ± 2.94 in B1to 39.27 ± 5.49 g/kg in B5.The fatty alcohols that are presentin sugarcane waxes are usuallyreferred to as policosanols, octacosanol being one of the most characteristicalcohols.p ≥ 0.05). On the other hand,when the filter cake was not dried before and after boiling with water, the concentration of phytosterols was higher than theother conditions. Interestingly, phytosterol contents in B5 extracts(10.99 ± 1.06 g/kg) were significantly lower than those in B6(31.56 ± 0.18 g/kg). The only difference between these methodswas that when using B5, the filter cake was dried prior to the boilingprocess, a higher time and energy-consuming method. Finally, principalcomponent analysis (PCA) of the data obtained by GC–MS  and to obtain higher extraction yield, the resultssuggest that the best boiling methods were the ones that includedacidic water boiling followed by isolation with ethanol (B5 and B6methods). Moreover, the procedure when the filter cake was dried priorto isolation  showed the highest content of 1-octacosanol,whose amount varied in samples as follows: B5 ≥ B2 = B4 ≥B6 = B1. In the case of coumaric acid, method B5 showed a trend towardhigher levels than when assaying B6 , but these differences were not statisticallysignificant , while samples obtained using B6 showed the lowest levels(3.45 ± 0.12 g/100 g). On the other hand, for this latter extract,B6, levels of triglycerides were significantly higher than those foundusing other conditions and its variation was as follows: 58.36 ±1.08 g/100 g lipids for B6 extract ≥ 38.37 ± 0.05 g/100g lipids for B5 extract ≥20.06 ± 0.48 g/100 g lipids forS extract ≥ 18.72 ± 0.92 g/100 g lipids for B4 extract≥14.28 ± 0.46 g/100 g lipids for B2 extract and ≥9.93± 1.64 g/100 g lipids for B1 extract.The isolation conditions of the different methodsgreatly affectedthe composition of the extract. As shown in 43 Aliphatic lipophilic compounds as tocopherols were previously quantifiedby del Río et al.44 in sugarcanestraw and bagasse extracts, which reported 70 mg/kg of the dry strawsample. Indeed, tocopherol was identified by HPLC-ELSD mainly usingthe B6 extraction protocol (3.25 ± 0.05 g/100 g lipids).As a result of theincreasing commercial interest in health-promotingfoods, tocopherol sources are widely studied.This analysis indicated that extracts obtained using the B6 methodhad a significantly lower concentration of glycolipids (3.45 ±0.12 g/100 g lipids) and a higher concentration of triglycerides (58.36± 1.08 g/100 g lipids). This can be observed in Overall, a distinct profile was observed by comparisonof all extractionprotocols. The used methodologies indicated that the B6 method wasmore efficient in extracting lipids such as fatty alcohols and phytosterolswhile maintaining lower total monosaccharide concentration. Hence,after analysis, the B6 method was found to be an efficient procedurefor extracting sugarcane filter cake lipids, designated from thispoint as waxes. Method B6 was used to extract wax from different sugarcanecrops, namely, Guariba (G) and Univalem (U), to explore the geographicallyrelated variation in the wax extract.Different batches of raw filter cake provided by Brazil  and collected in June 2020 were used to extract waxusing the B6 method due to its effectiveness in extracting fatty alcoholsand phytosterols while maintaining lower monosaccharide concentrationand avoiding energy-consuming steps . Extractsfrom Guariba and Univalem were labeled as B6_G and B6_U, respectively.p ≥ 0.05) were detected betweenthese two batches . By comparingB6_G and B6_U wax yields, it was possible to conclude that no significantdifferences .Total free fatty acids were quantified, and no significantdifferenceswere observed between batches . However, significantdifferences in the concentration of linoleic acid in B6_G (16.99 ±1.85 g/kg) and in B6_U (21.98 ± 0.34 g/kg) were found. The palmiticacid concentration was significantly lower in the B6_U batch (2.43± 0.11 g/kg) than that in B6_G concentration values of 1-octacosanol (18.51 ± 0.05 g/kg) thanB6_G (29.05 ± 1.87 g/kg). The same behavior was found in totalphytosterol content, which was higher (p < 0.05)for the B6_G (26.90 ± 0.07 g/kg) sample than B6_U (19.66 ±0.19 g/kg). In both samples, β-sitosterol was the main phytosterol,but campesterol  and stigmasterol  were also presentin these waxes.Fatty alcohols, mainly 1-octacosanol,were also identified in bothbatches. The B6_U batch presented lower (p <0.05) in the B6_G batch, being almost 3-fold the concentration foundin B6_U .Coumaric acid concentration was higher . The contentof triglycerides was also higher for B6_G (32.27 ± 0.52 g/100g lipids) than that for B6_U (24.27 ± 0.41 g/100 g lipids), andno major differences were found in the concentration of glycolipidsfor B6_G (11.68 ± 0.01 g/100 g lipids) when compared to B6_U(10.10 ± 0.05 g/100 g lipids).The results of the HPLC-ELSD analysisindicated that crops had significantly different tocopherol concentrations, 1712 cm–1 (−OH bending), 1168cm–1 (−C–O asymmetric stretching),and 1050–1035 cm–1 (−C–O stretching),which are usually associated with alcoholic functional groups, arefollowing the compositional GC–MS results that quantified fattyalcohols and phytosterols as two of the main families of compoundsthat constitute the analyzed waxes (B6_G and B6_U waxes).Samples (B6_G and B6_U waxes)were analyzed on a PerkinElmer Paragon 1000 FTIR with the ATR accessory.The spectra were obtained in the wavenumber range of 4000–550cm–1 arerelated to the C–H stretching of −CH2 groupsin aliphatic chains ; thevibrational bands at 1463 and 1377 cm–1 are associatedwith the C–H bending vibrations, respectively, of −CH2 and −CH3 groups, in aliphatic chains; andfinally, the identification of the vibrational bands at 730 and 719cm–1, both correlated to the rotational deformationof −CH2 groups in high aliphatic chains, indicatesthat the sample contains mostly aliphatic compounds of different functionalgroups, some of them with high aliphatic chains (≥C20). Moreover,these results were corroborated by the GC–MS analysis, whichidentified multiple aliphatic compounds within FFA, FOH, and aldehydes,namely, octacosanoic (C28) and triacontanoic (C30) acids, both withhigh aliphatic chains.Additionally,the vibrational bands at 2917 and 2849 cmFilter cake waxes’ (B6_G and B6_U)thermal properties werealso studied by differential scanning calorimetry (DSC) analysis 7. Both wStudies regarding antioxidant properties 8 and bio37 After determining the optimalextraction process and quantification of lipophilic compounds, theantioxidant activity using DPPH, ABTS, and ORAC assays was evaluatedfor B6_G and B6_U waxes, and the results are shown in It is established that sugarcane rind phytometabolitesthat areresponsible for its potential antioxidant activity can vary significantlyamong cultivated varieties. These compounds include anthocyanins,carotenoids, and terpenoids, which are positively correlated to sugarcaneantioxidant capacity.50 values were6.64 ± 0.16 and 6.07 ± 0.01 mg/mL for B6_G and B6_U, respectively than when using lower ethanol percentages.45 The antioxidant activity performed by DPPH  of sugarcane byproducts can range between 63 and1000 μg/mL, which is relatively higher than the obtained values.47 Furthermore, the total phenolic content in sugarcanemolasses extract was found to be 25.5 mg GAE/g elsewhere, which wassimilar to the obtained values in the current study.48The antioxidantactivity of sugarcane samples has been proved elsewhereand is correlated to the richness of these samples in flavonoids,phytosterols, and fatty alcohols.49The cytotoxicity of the B6 resulting waxes was testedon the humanimmortal keratinocyte cell line (HaCaT) to evaluate its biocompatibility.Hence, in 6 The determination of the lipophilic metaboliteprofile of sugarcane byproducts allows an extensive plant evaluationto predict its structural and metabolism-related functions.50Innovative applications for phytochemicalsderived through environmentallyfriendly extraction processes require cutting-edge separation andidentification methods based on multi-omics strategies.51 The antioxidant valuesobtained for the ABTS assay corroborated the pattern observed in theDPPH assay, where no significant differences occurred. Hence, theORAC values indicated that the Univalem batch presented higher antioxidantactivity. Overall, the crop’s location influenced the wax composition,but the antioxidant activity was uncompromised, as well as its thermalproperties. Hence, here, we demonstrate the suitability of wax productionfrom industrial wastes using the B6 method, independent of S. officinarum L. cultivar location and without influencingthe antioxidant performance. In fact, other studies related the richnessof sugarcane in phenolic compounds , as wellas in phytosterols , to its antioxidant,antimicrobial, and anti-inflammatory properties.52 Consequently, this research work suggests that lipid extractionprocedures do not require the usage of hazardous solvents, and a decreaseof sample drying steps as well as a 90% ethanol recovery was achieved,allowing to bypass higher energy requirements and accomplishing bettercompositional performance.Filter cake samples were subjected to different ethanoliclipidextraction protocols and varying sample pretreatments including pHvalue and drying steps, temperature, and sample/solvent proportion.By avoiding sample drying before boiling with water and afterwardethanol , the concentration of phytosterols was maximal.The obtained B6 extract yield was 9.59 ± 0.27 g/100 g of dryfilter cake, and concentrations of fatty alcohols and phytosterolswere 20.28 ± 1.48 and 31.56 ± 0.18 g/kg extract, respectively.Lower total monosaccharide concentration (26.19 ± 1.82 mg/g extract)was also the characteristic of this B6 resulting wax. This methodwas selected to extract wax from different sugarcane crops  and explore the geographically related variation inwax. Thus, the lowest obtained concentration in total monosaccharideswas found for the Univalem crop. Fatty alcohols, mainly 1-octacosanol,were also identified in all batches, but the Univalem batch presentedlower concentration values of 1-octacosanol than Guariba. In vitrofree radical scavenging assays were applied to access the potentialof sugarcane byproducts.


The FAKHRAVAC®, an inactivated SARS-CoV-2 vaccine, was assessed for safety and immunogenicity.In this double-blind, placebo-controlled, phase I trial, we randomly assigned 135 healthy adults between 18 and 55 to receive vaccine strengths of 5 or 10 μg/dose or placebo (adjuvant only) in 0–14 or 0–21 schedules. This trial was conducted in a single center in a community setting. The safety outcomes in this study were reactogenicity, local and systemic adverse reactions, abnormal laboratory findings, and Medically Attended Adverse Events (MAAE). Immunogenicity outcomes include serum neutralizing antibody activity and specific IgG antibody levels.The most frequent local adverse reaction was tenderness (28.9%), and the most frequent systemic adverse reaction was headache (9.6%). All adverse reactions were mild, occurred at a similar incidence in all six groups, and were resolved within a few days. In the 10-µg/dose vaccine group, the geometric mean ratio for neutralizing antibody titers at two weeks after the second injection compared to the placebo group was 9.03 (95% CI: 3.89–20.95) in the 0–14 schedule and 11.77 (95% CI: 2.77–49.94) in the 0–21 schedule. The corresponding figures for the 5-µg/dose group were 2.74 (1.2–6.28) and 5.2 (1.63–16.55). The highest seroconversion rate (four-fold increase) was related to the 10-µg/dose group .FAKHRAVAC® is safe and induces a strong humoral immune response to the SARS-CoV-2 virus at 10-µg/dose vaccine strength in adults aged 18–55. This vaccine strength was used for further assessment in the phase II trial.Trial registrationhttps://www.irct.ir; IRCT20210206050259N1.This study is registered with  Inactivated vaccines are among the oldest vaccine types developed against viruses. Their production and scale-up are relatively simple and low cost, and therefore, traditionally, they have been the first line of attempt by vaccine developers. In the SARS-Cov-2 pandemic, inactivated vaccines were among the first that received WHO emergency approval and were used on a wide scale FAKHRAVAC® is an inactivated SARS-Cov-2 vaccine. We used the G614 strain isolated from oropharynx swabs of confirmed SARS-CoV-2 patients admitted to Iranian hospitals for vaccine production. We replicated the virus using Vero cells (Cat. # 88020401), a WHO-approved cell line and inactivated it with formaldehyde. Aluminum hydroxide was used as the adjuvant at 1.5 mg/ml concentration 6 TCID50) and 10 μg/dose (2.5 × 106 TCID50) administered in two schedules of 0–14 and 0–21.FAKHRAVAC humoral immunogenicity and safety were assessed in four mammalian animals  in the preclinical phase We did a randomized, double-blind, placebo-controlled phase I clinical trial in healthy adults 18–55 years old. The first 15 people were enrolled in the study as sentinel subjects, and they were aware of the study groups they were assigned to (open-label). The allocation of the remaining 120 participants into 6 study groups was randomized and blind. There were two strengths of 5 and 10 µg/dose inactivated SARS-CoV-2 vaccine FAKHRAVAC® and placebo groups (receiving adjuvant-only preparation) administered in two schedules of 0–14 and 0–21. This trial was conducted in a single center in a community setting.This study was conducted in collaboration with the Iran University of Medical Science (IUMS) clinical trial center as the academic contract research organization (CRO). The study protocol was approved by the National Research Ethics Committee . Patients or the public were not involved in the design, or conduct, or reporting, or dissemination plans of our research.After approval by the Food and Drug organization, Iranian participants aged 18–55 years were invited to the study via a website. Participants who digitally signed (accepted) the informed consent form could complete the initial screening questionnaire online. Eligible candidates in the online screening were invited to attend the trial center, signed a paper informed consent, and further evaluated for eligibility. Inclusion criteria were: psychologically competent to volunteer for trial, age 18 to 55 years, body mass index between 18 and 35 kg/meter2, having complete health based on clinical and laboratory criteria, no current or previous SARS-CoV-2 disease, no pregnancy, negative IgG and IgM antibody titers against SARS-CoV-2 N antigen, negative RT-PCR test for SARS-CoV-2, IgG ELISA negative blood test against HIV, heart rate between 60 and 100, systolic blood pressure (between 90 and 140 mm Hg), diastolic blood pressure (between 60 and 90 mm Hg), use a safe method of contraception in men and women up to 3 months after the last injection of the vaccine. The main exclusion criteria were: positive RT-PCR test and presence of detectable antibody against SARS-CoV-2 nucleocapsid antigen; pregnancy; breastfeeding; history of syncope during injection or phlebotomy; history of immunodeficiency disorders (suspected or definite); long-term use of immunosuppressive drugs; history of allergic diseases such as angioedema or anaphylaxis; receiving chemotherapy or radiotherapy in the last five years; alcohol or drug abuse; any close contact with a person with confirmed SARS-CoV-2 in the two weeks leading up to the first vaccine injection. A complete list of eligibility criteria is available in the Study protocol (see S4 Protocol).This study utilized the variable size permuted block randomization method. The first 15 people entered the study gradually and in batches of 3 or 4 over nine days and were not blinded. The remaining 120 participants were randomly allocated into six groups, including a placebo group and two vaccine strengths of 5 and 10 µg/dose in two 0–14 and 0–21 schedules.The randomization codes were generated by the rand function and Excel software. Each participant was assigned a non-repeating four-digit number (randomization code) to conceal the randomization sequence. The study epidemiologist generated the random sequence.The identical vaccine and placebo single-injection vials were labeled by an independent team using the randomization codes. These codes were also embedded in the study software and successively assigned to the eligible participants. The 5 and 10 μg/dose strengths and placebo vials were the same shapes, color, and size. Thus, all participants, investigators, laboratory staff, and statisticians were unaware of the group allocations.After assessing vital signs and physical examination on day zero, each participant received the vaccine or the placebo based on the randomization sequence. Participants reported their vital signs and local  and systemic  adverse reactions during the seven days after each injection. All of the participants were invited to visit the research center for face to face assessment of safety and collecting blood samples for immunogenicity by study officers on days 7, 14, 21, 28, 42, 72, 90, 180, in the 0–14 schedule, and days 7, 14, 21, 28, 35, 49, 90, 180 in the 0–21 schedule. Follow-up at the second, fourth, and fifth-month intervals was conducted through phone calls (see S4 Protocol).The primary outcome was safety profile, including reactogenicity, local and systemic adverse reactions, and abnormal laboratory findings one week after injection. The secondary outcomes were serum neutralizing antibody activity measured by conventional virus neutralization test (for details of cVNT methodology please see the supplement 4) and specific IgG antibody levels, Medically Attended Adverse Events (MAAE), Serious Adverse Event (SAE), and Suspected Unexpected Serious Adverse Reactions (SUSAR).We asked the participants to report daily by filling out a self-administered questionnaire for seven days after injection and bringing it to the study center on their next visit. The research team made daily telephone contact with study participants during this period. All the items in the daily questionnaire were checked to ensure that they had been properly completed. Study participants could contact resident physicians at the center during the study period 24/7 if they needed any assistance or consultation and report any possible medical condition.Serum neutralizing antibody activity was assessed using a conventional virus neutralization test (cVNT). Specific IgG antibody against S antigen was measured by enzyme-linked immunosorbent assay (ELISA). We measured peripheral blood cytokine levels for IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-17, TNF-α, and γ-INF two weeks after the second injection to assess the inflammatory response. CD3, CD4, CD8, CD4/CD8, CD56, CD19 and CD20 were evaluated using flow cytometry.Patients and/or the public were not involved in this research's design, conduct, reporting, or dissemination plans.The sample size was 135 based on practical considerations and expert opinion. We assessed the safety outcomes in all participants who received at least one vaccine or placebo injection and the immunogenicity outcomes in all participants who received two vaccine or placebo injections. The primary approach for analysis was the intention to treat.Baseline comparisons were performed to examine the homogeneity between the study groups. For immunogenicity assessment, we calculated Geometric Mean Titers (GMT), Geometric Mean Ratio (GMR), Geometric Mean Fold Increase (GMFI), and their corresponding 95 % CIs based on a standard normal distribution of the log-transformations of antibody titers. A four-fold increase in antibody titers was considered seroconversion Between the 16th of March and the 13th of April 2021, 469 volunteers aged 18–55 years old were invited and screened for eligibility in phase I, of which 135 eligible participants were recruited into the trial. The first 15 participants were considered sentinel, and 120 remaining participants were randomly assigned to vaccine groups or placebo in two injection schedules of 0–14 and 0–21 .Fig. 1PaThe mean age of all 135 participants was 35 years , with 35 (25.9 %) individuals aged 18–30 years, 62 (45.9 %) aged 31–40 years, and 31 (23 %) aged 41–50 years, and 7 (5.2 %) aged 51 years or older across the groups. In this study, 69.6 % (94/135) of participants were male. Baseline characteristics of the participants were broadly similar across the groups .Table 1BThe safety findings show a favorable profile for this vaccine. No suspected unexpected serious adverse reactions (SUSAR) were observed in this study. The three patients with serious adverse events included hospitalizations due to SARS-CoV-2, lumbar disc herniation, and diagnostic curettage. All three were discharged in good general condition. Also, the only hospitalized patient due to SARS-CoV-2 belonged to the placebo group.In this study, 41.5 % (56/135) of participants reported at least one local adverse reaction seven days after the injection. The most common local adverse reaction was tenderness (28.9 %). Participants also reported pain (14.8 %), redness (3.7 %) and swelling/stiffness (2.2 %). In addition, 25.9 % (35/135) of participants reported at least one systemic adverse reaction within seven days after the injection. The most common reported systemic reaction was headache (9.6 %). Participants also reported Diarrhea (1.5 %), Fatigue(6.7 %), Myalgia(5.2 %), and None of the 135 participants reported Nausea/vomiting , Fig. 3.A total of 73 abnormal grade I and II laboratory results were observed in safety evaluation one week after the vaccination. One grade III lymphocytopenia was reported in a participant who received 10-µg/dose vaccine strength, and no grade IV was seen . All labThe serum neutralizing activity was undetectable (titer < 4) at baseline (before the first injection) in 134 of 135 participants. Two weeks after the second injection, participants in the 10 μg/dose group had a GMT of 9.03 (95 % CI: 5.17–15.8) in the 0–14 schedule and 11.77 (95 % CI: 2.29–60.52) in the 0–21 schedule. No increase in serum neutralizing activity was observed in the placebo groups. GMR and GMFI for 10 μg/dose group were 9.03 (95 % CI: 3.89–20.95) and 8.13 (95 % CI: 4.6–14.4) in the 0–14 schedule, and 11.77 (95 % CI: 2.77–49.94) and 8.43 (95 % CI: 0.78–91.56) in the 0–21 schedule, respectively. At 14 days after the second injection, the seroconversion (four-fold increase) rates of neutralizing antibodies were 71 % and 67 % in the 0–14 and 0–21 schedules, respectively .Table 2SThe inactivated FAKHRAVAC was tolerated well in all study groups. No vaccine-related serious adverse event was observed following the vaccination and subsequent follow-up period in any vaccine groups and administration schedules. Those who received two injections of the 10-µg/dose vaccine strength developed neutralizing antibody titers about nine times more than those who received the placebo two weeks after the second injection, and this was remarkably higher than the response in the 5-µg/dose vaccine recipients. The observed local and systemic reactions were generally mild and transient.Local and systemic adverse events were predominantly mild (grades I and II) and resolved within a few days. There was no clinically significant difference in the safety profile between the study groups, and the findings were similar to that of other SARS-CoV-2 vaccines We used fast protein liquid chromatography (FPLC) for vaccine purification, which is highly capable of separating host cell protein and host cell DNA impurities The vaccine elicited a safe immune response. Peripheral blood cytokine levels for IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-17, TNF-α, and γ-INF two weeks after the second injection showed no noticeable change from the baseline that reflects the ability of the immune system to self-contain the inflammatory process expected to be induced by vaccine injection . Furthermore, the flow cytometric examination of peripheral blood immune cell composition showed no considerable changes in the number and proportion of the cells. . Other studies on inactivated vaccines have also reported similar observations in their flow-cytometric and cytokine assessments in the peripheral blood We used a conventional virus neutralization test to assess specific antibody responses to SARS-COV-2 antigens. The test was conducted in a biosafety level III laboratory using a wild-type strain of the SARS-COV-2 virus isolated from the nasopharynx of Iranian hospitalized patients. Specific antibodies were also assessed by a commercially available kit (DiaZist®) and another kit developed in-house. However, we decided to draw conclusions based on the cVNT test results because of the inconsistencies in the results and questionable quality. As neutralizing antibody assessment was done by the same strain of the virus used for vaccine production and the SARS-COV-2 genome continues to change as a result of repeated mutations, we expect to lose some of the neutralizing power of FAKHRAVAC by the emergence of new strains of the virus. Therefore, in future use of this or any other SARS-COV-2 vaccine, updated COVID-19 vaccines based on prevailing strains will be needed.Our study had some limitations: First, because of ethical considerations due to inadequate antibody response, participants in the placebo and the 5-µg/dose groups were advised to enter the national SARS-CoV-2 vaccination program once it became available in late 2021. Therefore, some did not complete the six-month study follow-up period. Incomplete follow-up in the placebo group did not interfere with the dose selection decision, as it had been made following the interim analysis four weeks after the second injection. Second, we did not compare neutralizing antibody titers in our study with the convalescent serum samples as positive controls, which could have provided clues to the magnitude of the antibody responses. Third, the sample size in each group was relatively small as it is a common limitation among most phase I vaccine studies.FAKHRAVAC® at 10-µg/dose strength was safe and had a well-induced humoral immune response to the SARS-CoV-2 virus in adults aged 18–55 years in 0–14 and 0–21 schedules. It was selected for further clinical evaluation in a phase II clinical trial.Why was this study done?•Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused more than 573 million cases of SARS-CoV-2 worldwide, resulting in close to 6.4 million deaths so far.•We assessed the safety and immunogenicity of FAKHRAVAC inactivated SARS-Cov-2 in phase I clinical trialWhat did the researchers do and find?•Study participants received two strengths of 5 μg/dose (0.5 × 106 TCID50) and 10 μg/dose (2.5 × 106 TCID50) and placebo in two 0–14 and 0–21 administration schedules and underwent an intensive follow-up for six months.•Mild adverse reactions, such as pain, tenderness, and headache, were observed, and no severe adverse reaction was reported in all groups.•The 10 μg strengths of the vaccine delivered in the 0–14 schedule resulted in 9 fold increase in neutralizing antibody titer compared to the placebo group.•Administration schedules had little effect on efficacy.What do these findings mean?•FAKHRAVAC inactivated SARS-Cov-2 vaccine is safe and induces a strong immune response against SARS-CoV-2 disease.Akram Ansarifar: Conceptualization, Investigation, Validation, Writing – original draft. Ramin Hamidi Farahani: Methodology. Ahmad Karimi Rahjerdi: Project administration. Mohammadreza Ahi: Investigation, Validation, Writing – original draft. Ali Sheidaei: Data curation, Formal analysis, Investigation, Validation, Visualization, Writing – original draft. Kimiya Gohari: Data curation, Formal analysis, Investigation, Validation, Visualization, Writing – original draft. Zahra Rahimi: Investigation, Validation, Writing – original draft. Fatemeh Gholami: Investigation, Validation, Writing – original draft. Pouria Basiri: Investigation. Milad Moradi: Investigation. Arash Jahangiri: Investigation. Kosar Naderi: Project administration. Soheil Ghasemi: Investigation. Pezhman Khatami: Investigation. Mohsen Honari: Investigation. Samane Khodaverdloo: Investigation. Mohammad Shooshtari: Investigation. Hajar Mehr Azin: Investigation. Sohrab Moradi: Investigation. Batool Shafaghi: Investigation. Hossein Allahyari: Investigation. Arina Monazah: Investigation. Ali Khodaei Poor: Investigation. Hooman Bakhshande: Supervision. Zahra Taghva: Investigation. Mohammad Karimi Nia: Investigation. Masoud Solaymani Dodaran: Conceptualization, Investigation, Methodology, Project administration, Validation, Visualization, Writing – original draft. Mohsen Foroughizadeh: Conceptualization, Methodology.The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: SGH, PKH, MH, SKH, MSH, HM, SM, BSH, HA, AM, and AKH are employees of Milad Daro Noor Pharmaceutical (MDNP). MSD is an Iran University of Medical Science (IUMS) employee. MSD, AA, MA, ASH, ZR, FGH, and KG are members of the clinical trial center of the Iran University of Medical Science that acted as academic CRO. All other authors declare no other competing interests.