In vitro observation of macrophage polarization and gingival fibroblast behavior on three‐dimensional xenogeneic collagen matrixes

Abstract

Collagen biomaterials are widely used for soft tissue augmentation. Cross‐linking techniques for collagen matrix (CM) achieve mechanical and volumetric stability; nevertheless, cross‐linking may compromise biocompatibility. The aim of the present study was to investigate two different three‐dimensional (3D) porcine‐derived CMs, noncross‐linked (ncl)_CM and cross‐linked (cl)_CM, for their effects on macrophages (Mφ) and gingival fibroblasts. The effects of the CMs on the cell viability, proliferation, and polarization of Mφ derived from human monocyte THP‐1 cells were assessed. The effects of paracrine factors from Mφ cultured on the CMs were further studied in human gingival fibroblasts (HGF‐1 cells). The spongy layer of ncl_CM was partially resorbed after 1 day of culture. cl_CM maintained increased numbers of viable cells when compared with ncl_CM on day 3 for both THP‐1 and HGF‐1 cells. Higher mRNA levels of M1 markers, including IL‐1 and IL‐6, were found in Mφ cultured on cl_CM, while no significant differences were observed in M2 marker expression levels, including Arg1 and CD206, for cells cultured on both CMs when compared with those of the control. Furthermore, the conditioned medium collected from Mφ cultured on both CMs decreased cell viability. Nevertheless, neither of the CM‐conditioned media influenced the mRNA levels of TGF‐β, COL1a2, and PDGF‐A in HGF‐1 cells when compared with the control media. A comparison showed that cl_CM tended to result in more viable cells than ncl_CM, while cl_CM polarized Mφ toward an M1 phenotype, which was confirmed by the observation of increased mRNA levels of pro‐inflammatory cytokines.

In Vitro Comparison of Macrophage Polarization and Osteoblast Differentiation Potentials between Granules and Block Forms of Deproteinized Bovine Bone Mineral

“…Traditional methods, such as the paraffin embedding procedure, require decalcification and liquid handling, which destroys the fragile structure of cells cultured in vitro on materials. Moreover, Transwell ® inserts, whose membranes allow only liquid passage, successfully maintained granule stability during the liquid handling and the collection of samples in 24-well plates, as previously reported [17]. Different cell localization was clearly observed between DBBM granules and blocks by ALP staining of Saos-2 cells, showing a marked increase in ALP expression surrounding all DBBM granules.…”

“…THP-1-derived M0 Mϕs and Saos-2 cells were seeded on either DBBM granules or blocks at a density of 1.0 × 10 5 cells per insert, in 6.5 mm Transwell inserts with 0.4-µm pore polycarbonate membranes in 24-well plates. After 3 days for THP-1 cells, and after 14 days for Saos-2 cells, the cells were fixed in 4% paraformaldehyde for 15 min, and immediately frozen in Super Cryoembedding Medium (SCEM; Section-Lab, Hiroshima, Japan) in the stainless steel container as previously described [17]. Sections (10 µm) were cut by a cryomicrotome (Microm HM550, Thermo) with an adhesive film (Cryofilm Type 2C (16UF), Section-Lab) using Kawamoto's film method [18].…”

“…86, Sigma, St. Louis, MO, USA) as reported previously [19]. Briefly, the sections were further fixed in a citrate-acetone-formaldehyde fixative solution for 5 min, and then incubated in the alkaline dye mixture solution consisting of 1 mL of sodium nitrite solution and 1 mL of fast red violet alkaline solution, in 45 mL of distilled water and 1 mL of Naphthol AS-Bl alkaline solution, for 15 min, protected from light, at 37 • C. Immunocytochemistry was performed as previously described [17]. Briefly, after permeabilization with 0.2% Triton X-100 in Phosphate-buffered saline (PBS) for 10 min, the sections were blocked in 2% goat serum/PBS (Dako, Jena, Germany) for 1 h. Subsequently, the specimens were incubated overnight at 4 • C with primary antibodies against CD68 (Abcam, London, UK, dilution 1:100), CD86 (Santa Cruz, CA, USA, dilution 1:50), or CD206 (Santa Cruz, dilution 1:50).…”

Nanostructured Zirconia Surfaces Regulate Human Gingival Fibroblasts Behavior Through Differential Modulation of Macrophage Polarization

“…After taking the conditioned medium into consideration, the apoptotic rate of fibroblasts on TCP, BMZ, CSGZ, and SGZ surfaces reduced by 1.84, 3.67, 5.22, and 3.42%, respectively, compared with their respective controls (Figure 6), which indicated that CM collected from RAW264.7 culture in all samples slightly inhibited the apoptotic effect on fibroblasts. The conditioned medium from M2 macrophages has been shown to affect fibroblast behavior which secrete extracellular matrix (ECM), specifically collagen that can enhance efficacious implant integration (Fujioka-Kobayashi et al, 2020). Our Western blotting results showed that fibroblasts cultured with CM on BMZ surfaces produce more ECM with a higher expression of COL-I, VCL, and FN as compared with those on CSGZ and SGZ surfaces (Figures 8A-D).…”

Impact of Cross-Linking of Collagen Matrices on Tissue Regeneration in a Rabbit Calvarial Bone Defect

“…For instance, it was reported that the glutaraldehyde cross-linked collagen membranes demonstrated lower cell attachment and viability when compared to non-cross-linked collagen membranes in human gingival fibroblasts [9]. Nevertheless, our previous in vitro study proved the excellent cytocompatibility of the chemically cross-linked Cl_CM as well as Ncl_CM on human THP-1-derived macrophages Materials 2021, 14, 3740 2 of 11 and gingival fibroblasts [10]. However, slight immune cell responses were observed with the higher mRNA levels of M1 markers, including interleukin (IL)-1 and IL-6, in the macrophages cultured on Cl_CM [10].…”