The study is reported in accordance with the PRISMA 2020 guidelines. ²² The study protocol was registered on PROSPERO ( https://www.crd.york.ac.uk/prospero/#aboutpage), with registration number CRD42021252315 and was revised once as agreed upon by all authors. ²³

The eligibility criteria for inclusion of studies were defined using the PICOS criteria, Population (P) = PMMA heat polymerized or manufactured by CAD/CAM. Intervention (I) = Addition of TiO ₂NP in pure or hybrid form in PMMA. Studies based on TiO ₂ nanotubes, fillers, fibres and coating on PMMA were excluded. Control (C) = Heat polymerized PMMA without any addition of NPs or fibres. Outcome measure (O) = Antimicrobial properties, SR and SH of PMMA. Study designs (S) = In-Vivo and In-Vitro studies were included. Studies based on TiO ₂ nanotubes, fillers, fibres and coating on PMMA were excluded. Case-reports, systematic and narrative reviews, letters to the editor, short commentaries, pilot studies or studies with preliminary results and studies that were not related to the field of dentistry were excluded.

A comprehensive search strategy was developed for each database by an experienced librarian [C.P] and sent for peer reviewing using PRESS-Guidelines. ²⁴ Electronic searches in various databases were performed and reported using the PRISMA-S search extension wherein searches were done in databases, study registries, grey literature and other online sources. ²⁵ Based on the PRISMA-S guidelines, a rerun of the search was done with a gap of six months to search studies between January 2000 to April 2022, and carried out in three databases PubMed (NCBI), WHO Virtual Health Library and Cochrane Library. ²⁵ Two clinical trial registries ( Clinicaltrials.gov and Clinical Trials Registry of India), six other online sources (Ingenta Connect, Google Scholar, Research Gate, Dimension, Crossref and The Lens) and three sources for grey literature (BASE, OpenGrey and Grey Literature Report) were also searched. The PubMed search included both controlled vocabulary (Medical Subject Headings, or MeSH) and keyword terms for the concepts of “nanoparticles”, “titanium dioxide”, “PMMA”, “dentures”, “CAD/CAM”, “surface roughness”, “surface hardness”, “antibacterial” and “antifungal” and were used as the basis for all searches in other sources, with no usage of published search filters. Citation chasing and manual citation searching was performed of the reference lists of all the included articles by one researcher and E-mail alerts were set. The details of all the sources and their respective searches can be found in the (Supplementary Table 1, available online (Pragati, 2022)). De-duplication was performed using EndNote software.

Initially, two researchers (P.K. and A.H.) independently screened the titles and abstracts of the exported articles in Rayyan Software ( https://www.rayyan.ai/). Full-text articles were retrieved for potentially relevant articles and screened using the eligibility criteria by the same researchers. Wherever the full articles could not be retrieved, the corresponding authors were contacted, and the articles were obtained.

Antimicrobial Property was referred to as a collective term when there was an inhibition of the growth of bacteria and fungi, prevention of the formation of microbial colonies or destruction of microorganisms. Studies showing results of TiO ₂NP potential for the treatment of denture stomatitis were also included. Studies without a suitable control group, where the studies evaluated the antimicrobial capacity of the nanoparticle material by themselves, independently of their incorporation into PMMA were excluded. In case of disagreements, conflicts were resolved, however, articles in which the conflicts could not be resolved, the two reviewers (U.P. and M.J.) were consulted for the final decision. Two researchers (P.K. and A.H.) independently studied each of the included articles and extracted the data. (Supplementary Tables 1 and 2, available online (Pragati, 2022)) For data that was found to be missing, NS (not stated) was used and when not clear, ‘unclear’ was written.

The assessment of the quality of the included articles was performed using the modified Consolidated Standards of Reporting Trials (CONSORT) guidelines. ²⁶ The selected parameters were reported as “yes” or “no” on the screening of articles ( Table 1). The risk of bias was assessed individually by two researchers P.K. and A.H. and any disagreements were resolved by the third reviewer U.P. based on the modified Cochrane Risk of Bias tool and scored as described in previous study ²⁷ ( Table 2).

Due to the large methodological heterogeneity observed, narrative synthesis of the data was performed and quantitative analysis of the data was not performed.

Differences in the sample dimensions between the studies were observed, indicating methodological inconsistencies. Four studies mentioned the sample fabrications as per the recommended standardizations. ^{47 , 50 , 52 , 55} Even though the intent of the study was to include the effects of the addition of TiO ₂NP into CAD/CAM dentures, evidence on the comparison of these effects with conventional heat cured resins was found to be lacking. ^{37 , 42} Only one study used high impact acrylic resin and found that the hardness further increased by addition of 5 % TiO ₂NP, the difference being statistically significant. ⁴⁸ Despite the fact that polishing of the acrylic affects the surface properties and subsequent microbial adhesion, only seven studies were found to have mentioned the procedures adopted for finishing and polishing ^{44 , 45 , 49 , 51 , 53 , 58} and six studies mentioned details of sample storage. ^{45 , 46 , 48 , 52 , 54}

The methods of dispersion and surface treatments of NPs play a significant role in affecting the mechanical properties. NPs when treated with low molecular weight organosilicon surface treatment agents or silane coupling agents facilitate formation of a molecular bridge between the interface of the organic and inorganic substance. This results in increasing the NPs dispersion and enhanced the properties. ^{60 – 62} Even though the treatment can affect the resultant properties, only four studies mentioned the process of treating the NPs using silanization or any other form of surface treatment. ^{44 , 46 , 47 , 51} As surface treatment of the NPs has shown to improve properties, future studies must mention methods used for surface treatments.

Thorough mixing of NPs is essential as TiO ₂NP have high surface energies and agglomerate formation can act as stress concentrating centres thereby adversely affecting the properties. ^{52 , 57} Different methods of mixing and dispersion of the particles were used in the selected studies such as speed mixer, vacuum mixer, twin screw extruder, ultrasonication, planetary ball mill and ultrasonic homogeniser.

Considerable heterogeneity in methods of testing was observed in which researchers used methods such as antibacterial adhesion test, minimum inhibitory concentrations (MIC) of the NPs using microbroth dilution method and biofilm formation, MIC by broth culture, the plate count method were used. ^{45 , 47 , 51 , 53 , 55 , 57 , 58} Similarly, variation in the methods for testing SH was observed such as in Shore D hardness, ^{46 , 50} Vicker’s Hardness, ^{48 , 49 , 54} and Rockwell Hardness. ⁵⁶ While, SR was evaluated using a profilometer, ^{46 , 50 , 52} perthometer, ⁵⁸ and digital microscope. ⁵¹

The results of the current study indicate that the size of the added TiO ₂NP did not affect the observed antimicrobial effect and SH as studies that used smaller TiO ₂NP (less than 50 nm) ^{52 , 53 , 55 , 57 , 58} as well as those that used relatively larger sized NPs (greater than 50 nm), ^{45 , 51} reported improved antimicrobial properties. Previous studies have found that smaller sized NPs improve properties as they can penetrate between linear chains and form homogenous mixture, and additionally can easily attach and penetrate cell membranes. ^{63 – 65} Although studies reported conflicting results regarding the effect on SR, SH was found to increase irrespective of the size of TiO ₂NP added to PMMA. In-order to provide stronger evidence on the optimum size of NPs to achieve improved properties, future studies must justify selection of a size as well as analyse the effect of increasing or decreasing the size of NPs on the given properties.

Previous studies have found that a smaller percentage of the added NPs ensures that the NPs are well embedded into the resin. ^{66 , 67} From the analysis of the obtained results it was found that increasing the percentage of addition of TiO ₂NP did not always improve the antimicrobial effect. The most promising results were obtained with 3% of addition of TiO ₂NP, as four studies demonstrated improved antimicrobial effect with 3% TiO ₂NP. ^{45 , 47 , 51 , 55} The observed antimicrobial effects can be attributed to the different mechanisms of action such as the ability of TiO ₂NP to produce cytotoxic oxygen radicals, reduction in the bacterial attachment and biofilm formation onto polymer surfaces and deactivation of cellular enzymes causing to cell death. ^{47 , 68 – 72}

Regarding the effect on SH, the current analysis found that although with an increase in the percentage of TiO ₂NP, the SH of the nanocomposite increases, little or no improvement occurred with percentages below 3%. The variations in the SH are said to be due to the dispersion of the NPs other factors such as use of silane coupling agent, mixing methods used for dispersion for minimum agglomerates formation and the greater presence of cross-linking in the surface layers than the internal layers of the material. ^{45 , 73 – 75}

With regards to the SR, the conclusive effect of increasing the percentage could not be obtained, however, at 3% the SR was found to increase. Factors such as the dispersion of the NP to close the gap in the resin matrix, presence of the amount of nanoparticle on the surface and hydrophobicity of the nano-resin composite play important roles to determine the SR. ^{76 – 78} The clinical significance of this property cannot be underestimated as with the increase in the SR larger number of sites are available for microbial adhesion, however only two studies evaluated the microbial adhesion on addition of different concentrations of TiO ₂NP. ^{18 , 51 , 58 , 79}

The current review differs from previous reviews as the effect of nanohybrid combination using TiO ₂NP was also evaluated, wherein it was observed that the nanohybrid combination of TiO ₂ and SiO ₂ showed antimicrobial action against Streptococcus M. ⁵⁷ Studies have found that TiO ₂NP on doping with other metals or metal oxides, showed improved photocatalytic activity thereby showing improved antimicrobial properties. ^{80 , 81} Keeping in view with the limited available evidence but with promising results, future research on analysing combination of metal oxide NPs with TiO ₂NP can be undertaken.

Based on the results of the current systematic review, the use of TiO ₂NP size up to 100 nm and at 3% concentration can be recommended to achieve optimum antimicrobial effect, SH and SR, however, additional efforts must be undertaken to improve the SR so as to minimize the microbial adhesion. Future studies can be undertaken to assess the impact to different methods to improve the SR with their impact on the microbial adhesion.

The current study has certain limitations. Firstly, due to the significant diversity within the primary studies, meta-analysis of the results could not be performed and the conclusions are drawn based on narrative synthesis. Secondly, the results of the current review must be interpreted with careful understanding as included studies showed moderate results in methodological and risk assessments due to the in-vitro nature of the majority of the studies, where uniform guidelines are currently missing. ^{82 , 83} The strength of the current study is the use robust methodology and use of a comprehensive methodology of a literature search based on the recently introduced PRISMA-S guidelines, minimizing the publication bias and following a robust methodology in the conduction of the systematic review and provides updated information on the use of the pure form and hybrid TiO ₂NP.