1. IntroductionProsthetic restorations using dental implants have increased in popularity and prevalence [1]. However, the occurrence of peri-implant complications such as peri-implant mucositis and peri-implantitis is rapidly increasing [2]. For dental implant restoration, the prosthesis must extend from the implant platform through the connective tissue and junctional epithelium into the oral cavity. Depending on the implant position, tissue healing, and prosthetic strategy, the alveolar bone can be in close proximity to provisional restorations. Ideally, the materials used for this transmucosal portion should be nontoxic to the contacting and adjacent tissues and should not induce peri-implant gingival recession and bone loss [3,4,5,6]. Preferably, the materials should allow peri-implant connective tissue to adhere and form a stable physical barrier to prevent the invasion of oral bacteria, which could jeopardize osseointegration and implant longevity [4,7]. Thus, prior to delivery of the definitive implant restoration, provisional implant restorations play important roles in establishing anatomical and biophysiological stability and a soft tissue seal during the critical stage of healing [8,9].Various materials such as self-curing poly (methyl methacrylate) (PMMA) acrylics, milled PMMA acrylics, bis-acrylics, and composite resins are used in implant provisional restorations [10,11,12]. Self-curing acrylics are used on demand [13,14,15], while milled PMMA acrylics are made from a PMMA block/disc prepolymerized at a high temperature and pressure and subsequently machined using computer-aided design/computer-aided manufacturing (CAD/CAM) systems. Unlike PMMA, bis-acrylics contain filler particles and shrink less after polymerization. Composite resins are made of inorganic fillers, photoinitiators, and matrix monomers such as bisphenol A glycidyl methacrylate (bis-GMA) and urethane dimethacrylate (UDMA).The chemical composition of polymer-based materials might alter the biological properties and responses of the peri-implant soft tissue. A number of studies have reported the cytotoxicity of acrylics, which is variable due to the chemical composition, type, and quantity of the leaching residual monomer [16,17,18,19,20,21,22,23,24,25,26,27,28,29,30]. Polymer-based materials also generate free radicals during and after polymerization, which cause significant cellular damage [19,20,26,31]. Conversely, prefabricated PMMA blocks are assumed to produce minimal or no residual monomers or free radicals. Despite emerging theoretical knowledge about the properties and effects of these materials, the choice of material remains largely based on operator preference with limited consideration of their biocompatibility, most likely due to a lack of robust, systematic, and fundamental data to inform clinical decision-making.While there have been several studies of cellular responses to definitive implant restoration materials, there is less data on the commonly used provisional restoration materials [12,16,32,33]. More importantly, a very limited number of studies have examined the effects of provisional materials on multiple cell types simultaneously [34]. This is important since the biocompatibility of provisional materials is likely to influence both soft and hard tissue cells in different locations, so care must also be taken to consider the positional relationship between the cells and material to mimic the clinical context. Therefore, the objectives of this study were to (i) evaluate the behavior and function of human oral fibroblasts and osteoblasts in the presence of five different provisional dental restoration materials including titanium (Ti) alloy as a bioinert control, and (ii) compare responses of the two different cell types to determine potential material-cell type crossover modulation. Cellular behavior and function were separately examined for cells in direct contact with, and in close proximity to, the materials to mimic the intraoral environment.

We hypothesize that the biocompatibility of implant provisional materials varies more significantly than we anticipate in daily clinical practice and that the adverse effect of selected materials remains significant even in proximity, without direct contact. We also postulate that osteoblasts, which are categorized into differentiating cells, are more susceptible to material toxicity than nondifferentiating gingival fibroblasts. The would-be-obtained results in this study will provide a foundation for understanding and selecting various materials during implant provisional restoration.

2. Materials and Methods 2.1. Material Preparation and CharacterizationFive different test materials in rectangular plate form (6 mm × 14 mm, 2 mm thick) were prepared (Figure 1A, Table 1). Bis-acrylic, composite, and self-curing acrylic were prepared using standardized silicone molds prepared for each material and according to the manufacturer’s instructions. Milled acrylic plates were designed using CAD software (123D Design, Hyperdent®, Synergy Health, Sydney, Australia) and machined from PMMA disks with a milling machine (Versamill 5 × 200, Axsys Dental Solutions, Wixom, MI, USA). Acrylic plates were washed with a steam cleaner and disinfected with 75% ethanol. Machined Ti alloy plates were manufactured as a positive control. Surface topography was examined by scanning electron microscopy (SEM; Nova 230 Nano SEM, FEI, Hillsboro, OR, USA). 2.2. Cell Culture

Human gingival fibroblasts were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA) and grown in a fibroblast medium supplemented with 5% fetal bovine serum (FBS), 1% fibroblast growth supplement-2, and 1% penicillin/streptomycin solution. Immortalized human bone marrow mesenchymal stromal cells (hMSCs) were purchased from Applied Biological Materials Inc. (Richmond, VC, Canada) and grown in alpha-modified Eagle’s medium (αMEM) supplemented with 15% FBS and 1% antibiotic-antimycotic solution. Cells were incubated in 95% air and 5% CO2 at 37 °C. The medium was changed every three days, and the cells were passaged with 0.05% trypsin-EDTA at 80% confluence. Cells at passages 5–8 were seeded at a density of 4 × 104 cells/well onto each test material placed in a well (20 mm diameter) of 12-well culture plates. An osteogenic induction medium consisting of the growth medium with 100 nM dexamethasone, 10 mM sodium β-glycerophosphate, and 0.05 mM ascorbic acid was used from the time of seeding. Therefore, the cells were defined and termed as osteoblasts from the onset of seeding in the present study. The UCLA Institutional Biosafety Committee approved the study protocol (BUA-2-22-036-001).

2.3. Quantification of Attached and Propagated CellsThe number of attached cells was counted to determine the “contact” effect and the “proximity” effect, where the contact effect was the quantification of cells attached directly to the test materials and the proximity effect was the quantification of cells attached to the well of the culture dish around the materials (Figure 1B). Water-soluble tetrazolium salt (WST-1)-based colorimetric assays were used to quantify cell viability. Attached cells were measured two days after seeding, while propagated cells were measured four and six days after seeding. The amount of formazan product was measured at an absorbance of 450 nm using a microplate reader (Synergy H1, BioTek Instruments, Winooski, VT, USA). 2.4. Fluorescence Microscopy

Cell morphology was visualized four days after seeding by fluorescence microscopy (DMI6000B, Leica Microsystems, Wetzlar, Germany). Cells were dual stained with DAPI to visualize nuclei and rhodamine-phalloidin to visualize actin filaments. Cell density was quantified on each test material by counting cells visualized in the images.

2.5. Collagen Production

Fibroblast collagen production was quantified with a soluble collagen assay (ab241015 Soluble Collagen Assay Kit, Abcam, Cambridge, UK). Four days after seeding, cells were manually detached and collected in PBS, pelleted by centrifugation, and 0.5 M acetic acid was added to the cell pellet. Soluble collagen in acetic acid, followed by enzymatic degradation of collagen into glycine-rich oligopeptides, was quantified using a fluorogenic reagent and developer solution that selectively reacts with N-terminal glycine fragments to form a stable fluorescent complex (Ex/Em = 376/468 nm).

2.6. Alkaline Phosphatase (ALP) Activity

Osteoblast ALP activity was examined on day four using a colorimetry-based assay. After removing the samples from the culture wells, the wells were rinsed with double-distilled water (ddH2O) and treated with 250 µL p-nitrophenyl phosphate before further incubation at 37 °C for 15 min. ALP activity was evaluated as the amount of nitrophenol released through the enzyme reaction and measured at an absorbance of 405 nm using a microplate reader.

2.7. Compatibility Index for Fibroblasts and Osteoblasts

To quantify which cell type was more susceptible to the toxicity of each material, a compatibility index was calculated by dividing the higher number of fibroblasts or osteoblasts by the lower number of fibroblasts or osteoblasts. If the numerator was the number of fibroblasts, the material provided a more favorable, profibroblastic environment, and vice versa. The index score was calculated relative to the Ti alloy.

Using a WST-1 value for the contact effect of the self-curing acrylic on day 2 as an example, the compatibility index was expressed as (WST-1 value of fibroblasts on self-curing acrylic/WST-1 value of fibroblasts on titanium alloy)/(WST-1 value of osteoblasts on self-curing acrylic/WST-1 value of osteoblasts on titanium alloy).

2.8. Statistical Analysis

Results are expressed as means ± standard deviations from triplicate experiments (n = 3). The five materials were compared with a one-way analysis of variance (ANOVA) with Bonferroni post hoc correction. Two-way ANOVA was performed to evaluate differences between the test materials at varying time points. Two groups were compared using Student’s t-test. Any p-values less than 0.05 (alpha value of 0.05 and confidence level of 0.95) were deemed statistically significant.

4. Discussion

Here we comprehensively assessed the biocompatibility of implant provisional restoration materials with human gingival fibroblasts and osteoblasts by evaluating cellular behavior and function on and around the materials. This allowed us to determine whether the biocompatibility varied for different cell types and whether it differed when cells were in direct contact or adjacent to the potentially cytotoxic materials.

This study implied that implant provisional restoration materials may adversely affect soft tissue healing and osseointegration depending on the material. In culture experiments, fibroblasts and osteoblasts can either adhere to materials or around the materials, with those that do not attach undergoing cell death. Therefore, quantifying the number of cells attaching to either the test materials or the culture wells represents an indirect measurement of cell survival in the face of material cytotoxicity. Here, we tested four representative provisional restoration materials and Ti alloy, the latter chosen as a positive control because it is known to be bioinert and widely used for temporary abutments [35,36,37,38,39,40]. The number of cells attaching to a test material was regarded as a contact effect, while the number of cells attaching to the well around the test material was regarded as a proximity effect. We found that initial fibroblast and osteoblast settlement/attachment and subsequent propagation varied considerably ranging from lethal and tolerant to inert, depending on the material tested. As expected, the highest number of cells attached and propagated on the Ti alloy at all time points. Unexpectedly, osteoblasts, but not fibroblasts, attached to the composite. Compared with Ti alloy, fewer osteoblasts attached to the self-curing acrylic and milled acrylic than fibroblasts. However, the milled acrylic showed close cytocompatibility to the Ti alloy and the self-curing acrylic was significantly less cytotoxic than bis-acrylic and composite. As expected, attachment and proliferation improved slightly in proximity experiments, suggesting that there was less of a chemical effect exerted on cells in close proximity to the materials. Surprisingly, only a few osteoblasts attached around bis-acrylic. Of clinical note, the fibroblasts and osteoblasts had differing susceptibilities to the cytotoxic effects of the tested materials.Some studies have shown that the cytotoxicity of materials depends on their constituents including monomers, polymerization initiators, and filler particles [32,41,42,43]. Unreacted monomers have critical cellular effects [16,30]. Bis-GMA and UDMA are the main ingredients of the composite, and bis-GMA is eluted at higher concentrations than UDMA [44]. Both cause DNA strand breaks in fibroblasts, which would account for the observed cytotoxicity [45]. Our results suggest that bis-GMA and UDMA are more toxic than MMA, while UDMA is less toxic than bis-GMA, especially to fibroblasts. Monomers do not appear to be eluted from milled acrylic [46], and our results confirm that milled acrylic is more cytocompatible than self-curing acrylic, most likely due to the lower residual monomer composition.Benzoyl peroxide (BPO) and camphorquinone (CQ) are major polymerization initiators that can also compromise cell viability. BPO is an initiator for the self-curing acrylic resin that is broken down during polymerization to release radicals that injure adjacent cells [17,23,24,28,31]. CQ is a well-known photoinitiator for composite resins that promotes polymerization by generating free radicals with an amine as a coinitiator [47]. Our results show that self-curing acrylic is less cytotoxic than composite, suggesting that BPO is likely to be less cytotoxic than CQ.

Collagen production was generally proportional to the number of cells, but the production of collagen around the self-curing acrylic was disproportionately higher than around the other materials. This means that self-curing acrylic is less deleterious to fibroblast function. In addition, the milled acrylic had no toxic effect on collagen production due to the much lower residual monomer composition than self-curing acrylic.

Osteoblasts growing on the Ti alloy had the highest ALP activity, followed by those on milled acrylic. The ALP activity of cells growing around the self-curing acrylic was similar to that around milled acrylic, so self-curing acrylic may have little deleterious effect on osteoblast function. Despite some osteoblasts attaching and propagating on the composite, there was no detectable ALP activity. There was also a time-dependent decrease in the number of osteoblasts growing on bis-acrylic and composite, highlighting persistent toxicity resulting in compromised osteoblast function for both materials.

It is known that the surface topography of biomaterials influences the initial cellular behavior [48,49,50,51,52]. In theory, the smoother the surface, the better the cell attachment and proliferation [12,51,53,54,55,56,57,58]. Although the primary focus of this study was the chemical effect of materials on cellular activity, qualitative analysis of the surface morphology by SEM provided some useful insights into the cell-material interactions. For example, cell attachment and proliferation were higher on materials with relatively smooth surfaces (self-curing acrylic, milled acrylic, and Ti alloy), consistent with the hypothesis that smooth surfaces promote attachment and proliferation. These trends were especially prominent for osteoblasts. Based on these insights and hypothesis, future studies are required to quantitatively assess the surface morphology of the test materials including the average roughness and the peak-to-valley roughness to explore the contribution of topography/roughness factors in determining the biocompatibility of implant provisional materials.

The hybrid assessment of contact and proximity effects was designed to mimic the local environment of peri-implant tissue, i.e., the connective tissue and alveolar bone, both of which are exposed directly and indirectly to provisional materials. The optimal environment for fibroblasts and osteoblasts was indicated by our compatibility index, which varied according to the assay. Self-curing and milled acrylic were pro-fibroblastic at all time points, with self-curing acrylic showing the highest index and milled acrylic approaching a value of one due to later osteoblastic plateauing.

Conversely, results were different in proximity experiments, with bis-acrylic and composite indices pro-osteoblastic. Milled acrylic was slightly pro-osteoblastic at all time points. In another study examining the cytotoxicity of different surface treatments of composite, there was a trend towards a pro-osteoblastic phenotype [59], consistent with our results. We separately evaluated the contact and proximity effects of each material for fibroblasts and osteoblasts and provided robust, fundamental data, which warrants further studies such as in vitro mechanistic studies and animal studies.