Table 7.

Bioactive coatings: in vitro experiments.

Substrate	Coating Material	Control	Cells	Test Performed	Main Results	References
CpTi	Sphene	Uncoated	hADSCs	- MTT assay - SEM analysis - Immunofluorescence - Alzarin Red S staining - rt-PCR	Sphene-based coating significantly better supported cell attachment and proliferation, than CpTi samples. When cells were seeded in the presence of osteogenic differentiation medium for 21 days, a significantly higher accumulation of calcium deposits on sphene coatings than on uncoated samples was observed.	[37]
Ti-6Al-4V	Sphene	Uncoated	Primary human osteoblasts	- SEM analysis - MTS assay - rt-PCR	After seven days of culture, cell proliferation rate on hardystonite coatings was higher when compared with those on sphene coatings and Ti-6Al-4V samples (p < 0.05). Both coatings were able to enhance the expression of bone-related genes.	[135]
	Hardystonite
Ti-6Al-4V	Sphene	HA-coated (Uncoated)	Human osteoblast-like cells	- SEM analysis - MTS assay - ICP-AES - ALP activity	- After seven days of culture, significantly higher cell proliferation and ALP activity on sphene coatings than on HA-coated and uncoated substrates were observed (p < 0.05). After seven days of culture, no detectable levels of Ti ions and minor amounts of Ca and Si ions released from sphene coatings.	[127]
Ti-6Al-4V	Hardystonite	Uncoated	MC3T3-E1 cells (a mouse calvaria-derived osteoblast-like cell line)	- SEM analysis - MTS assay	Hardystonite showed no toxic effect on cells. After 24 h incubation, cells on hardystonite coating were more elongated, spread and confluent than on uncoated samples.	[139]
Ti-6Al-4V	Hardystonite	HA-coated	Canine BMMSCs	- Immunofluorescence - rt-PCR - ICP-OES - ALP activity - Calcium deposition assay	After 14 days of culture, the expression levels for BMP-2, ALP and osteocalcin cells cultured on strontium-substituted hardystonite coatings were the highest, followed by hardystonite and then by HA coatings.	[140]
	Sr-substituted hardystonite
Ti-6Al-4V	Akermanite	HA-coated	Rabbit BMMSCs	- SEM analysis - MTT assay	After one day, cells on HA coating were similar in appearance to those on akermanite coating, but with fewer minor filopodia. After seven days of culture, more cells were detected on the akermanite coating than on the HA one. After one day of culture no significant differences in cell proliferation rate between the two groups; cells on the akermanite coatings showed a higher proliferation rate than that on HA coatings at both three and seven days of culture (p < 0.05 and p < 0.01, respectively).	[141]
Ti-6Al-4V	Bredigite	- HA-coated - Blank control	Rabbit BMMSCs	- SEM analysis - MTT assay	Cells cultured on bredigite coating for one day presented an elongated morphology and were firmly attached to the surface. After three days of culture, the bredigite coating presented numerous cells on its surface, characterized by a net-like morphology. After three and seven days of culture, cells on bredigite coating had a higher proliferation rate than that on HA coating and blank control.	[143]
Ti-6Al-4V	Wollastonite	–	Rat BMMSCs	- MTT assay - ICP-AES - Immunofluorescence - ALP activity - qRT-PCR	Cells seeded on the HT treated coatings presented higher cell viability and proliferation than untreated coatings at all time points (one, four and seven days) (p < 0.05). Quantitative results of ALP activity cells cultured on HT treated and untreated coatings revealed higher ALP activity on HT treated samples at all time points (four, seven and 10 days) (p < 0.05). HT treatment enhanced the expression of osteogenic genes and angiogenic factors.	[53]
CpTi	45S5 Bioglass	Uncoated	Human osteosarcoma cell line MG63	- MTT assay - SEM analysis	After 24 h of culture, cells spread over the coated surface. After seven days, it appeared covered by a cell layer. Coated samples supported an increasing cell viability overtime, similarly to uncoated samples.	[150]
Ti grade 4	x CaO·(1 − x)SiO2 bioactive glass (0.0 ≤ x ≤ 0.60)	Uncoated	NIH 3 T3 murine fibroblasts cells	- WST-8 assay	After 24 h of culture, the cells grown on uncoated samples showed lower viability than on all coated samples (p < 0.05). The best results were obtained with 0.3CaO·SiO2 and 0.4CaO·SiO2 coatings, which were homogeneous and crack-free, contrary to SiO2, 0.5CaO·SiO2 and 0.6CaO·SiO2 coatings.	[1]
Ti grade 4	70S30CxA bioactive glass (in mol%: 70% SiO2 (S), 30% CaO (C), x% Ag2O (A), with 0.08 ≤ x ≤ 0.27	Uncoated	NIH 3 T3 murine fibroblasts cells	- WST-8 assay	Higher percentage of viable cells on coated samples than on uncoated ones. The coating with the lower content of Ag resulted to be the most biocompatible.	[154]
Mg alloy (AZ91)	Diopside + bredigite + fluoridated HA	(a) Uncoated; (b) ASD coated	L-929 fibroblast cell line	- MTT assay - SEM analysis	Increase in cell viability from two to seven days of culture in all samples. At all time points (two, five and seven days) cell viability was as follows: diopside + bredigite + fluoridated HA coated > ASD coated > uncoated.	[66]
Mg alloy (AZ91)	Diopside	(a) Uncoated; (b) MAO coated	L-929 fibroblast cell line	- MTT assay	Cell viability of all samples increased with the culture time. At all time points (two, five and seven days) cell viability was as follows: diopside coated > MAO coated > uncoated. Diopside coated samples had significantly higher cell viability than that of uncoated samples at all time intervals (p < 0.05).	[65]
Mg alloy (ZK60)	Dimagnesium silicate – Magnesium oxide	Uncoated	Human osteoblast-like cells (MG63) and NIH 3 T3 murine fibroblasts cells	- CellTiter-96 cytotoxicity test - SEM analysis	Dimagnesium silicate-magnesium oxide coatings, with or without gallic acid, favored osteoblast-like cell proliferation.	[11]

ALP = alkaline phosphatase; BMMSCs = bone marrow mesenchymal stem cells; BMP = bone morphogenic protein; hADSCs = human adipose-derived stem cells; qRT-PCR = quantitative reverse transcription polymerase chain reaction; rt-PCR = real time polymerase chain reaction.