Cell Type-Specific Effects of Implant Provisional Restoration Materials on the Growth and Function of Human Fibroblasts and Osteoblasts

Abstract

Implant provisional restorations should ideally be nontoxic to the contacting and adjacent tissues, create anatomical and biophysiological stability, and establish a soft tissue seal through interactions between prosthesis, soft tissue, and alveolar bone. However, there is a lack of robust, systematic, and fundamental data to inform clinical decision making. Here we systematically explored the biocompatibility of fibroblasts and osteoblasts in direct contact with, or close proximity to, provisional restoration materials. Human gingival fibroblasts and osteoblasts were cultured on the "contact" effect and around the "proximity" effect with various provisional materials: bis-acrylic, composite, self-curing acrylic, and milled acrylic, with titanium alloy as a bioinert control. The number of fibroblasts and osteoblasts surviving and attaching to and around the materials varied considerably depending on the material, with milled acrylic the most biocompatible and similar to titanium alloy, followed by self-curing acrylic and little to no attachment on or around bis-acrylic and composite materials. Milled and self-curing acrylics similarly favored subsequent cellular proliferation and physiological functions such as collagen production in fibroblasts and alkaline phosphatase activity in osteoblasts. Neither fibroblasts nor osteoblasts showed a functional phenotype when cultured with bis-acrylic or composite. By calculating a biocompatibility index for each material, we established that fibroblasts were more resistant to the cytotoxicity induced by most materials in direct contact, however, the osteoblasts were more resistant when the materials were in close proximity. In conclusion, there was a wide variation in the cytotoxicity of implant provisional restoration materials ranging from lethal and tolerant to near inert, and this cytotoxicity may be received differently between the different cell types and depending on their physical interrelationships.

Keywords: cytotoxicity; fibroblast; osteoblast; peri-implant tissue; provisional restoration.

Figure 1

Test materials and the experimental design for counting cells. (A) Prepared rectangular samples (6 mm × 14 mm, 2 mm thick). a, bis-acrylic; b, composite; c, self-curing acrylic; d, milled acrylic; and e, Ti alloy. (B) Attached cells were counted to determine contact and proximity effects, where the contact effect was the quantification of cells attached to test materials and the proximity effect was the quantification of cells attached to the well of the culture dish (20 mm diameter) around the materials.

Figure 2

Surface topography of the test materials by scanning electron microscopy (SEM). (A) Low-magnification SEM images (×1000). (B) High-magnification SEM images (×10,000).

Figure 3

Successful attachment of fibroblasts and osteoblasts during initial culture (day 2). (A) The number of attached fibroblasts on test materials (contact effect). (B) The number of fibroblasts attached to the culture dish around the test materials (proximity effect). (C) The number of attached osteoblasts in contact experiments and (D) in proximity experiments. Data shown are mean ± SD. Significant differences are shown (one-way ANOVA with the Bonferroni post hoc test, * p < 0.05).

Figure 4

Cell propagation after initial settlement (days 4 and 6). (A) The number of propagated cells on test materials. (B) The number of propagated cells around test materials. (C) The number of propagated osteoblasts on test materials. (D) The number of propagated osteoblasts around test materials. Data shown are means ± SD (n = 3). Significant differences are shown (Student’s t-test, * p < 0.05).

Figure 5

Visualization of fibroblasts and osteoblasts on test materials 4 days after seeding. (A) Fibroblasts were dual stained with DAPI for nuclei and rhodamine-phalloidin for actin filaments. (B) The number of fibroblasts in these images was counted to confirm the result in Figure 3A. (C) Osteoblasts stained similarly to fibroblasts. (D) The number of osteoblasts in these images was counted to confirm the result in Figure 3C. Data shown are means ± SD (n = 3). Significant differences are shown (one-way ANOVA with the Bonferroni post hoc test, * p < 0.05).

Figure 6

Collagen production by fibroblasts 4 days after seeding. (A) Collagen production on test materials and (B) around test materials. Data shown are means ± SD (n = 3). Significant differences are shown (one-way ANOVA with Bonferroni post hoc test, * p < 0.05).

Figure 7

Alkaline phosphatase (ALP) activity of osteoblasts 4 days after seeding. (A) ALP activity on test materials and (B) around test materials. Data shown are means ± SD (n = 3).Significant differences are shown (one-way ANOVA with Bonferroni Post hoc test, *p < 0.05).

Figure 8

Favorable environments for fibroblasts and osteoblasts compared with Ti alloy. The compatibility index represents the higher number of fibroblasts or osteoblasts divided by the lower number. If the numerator was the number of fibroblasts, the material provided a more favorable, pro-fibroblastic environment. The index score was relative to Ti alloy. (A) Compatibility index related to the contact effect and (B) the proximity effect. Abbreviation: N/A, not applicable.