Proteome analysis of human mesenchymal stem cells undergoing chondrogenesis when exposed to the products of various magnesium-based materials degradation

Abstract

Treatment of physeal fractures (15%–30% of all paediatric fractures) remains a challenge as in approximately 10% of the cases, significant growth disturbance may occur. Bioresorbable Magnesium-based implants represent a strategy to minimize damage (i.e., load support until bone healing without second surgery). Nevertheless, the absence of harmful effects of magnesium-implants and their degradation products on the growth plate should be confirmed. Here, the proteome of human mesenchymal stem cells undergoing chondrogenesis was evaluated when exposed to the products of various Magnesium-based materials degradation. The results of this study indicate that the materials induced regulation of proteins associated with cell chondrogenesis and cartilage formation, which should be beneficial for cartilage regeneration.

Graphical abstract

Highlights

• •Degradation products from Mg-based materials generated changes in protein expression.
• •Relevant proteins involved in cartilage formation were upregulated.
• •Potential application of especially Pure-Mg and Mg-10Gd for cartilage regeneration.

1. Introduction

Biodegradable magnesium (Mg)-based materials are promising candidates for substituting permanent implants for orthopaedic application as a second surgery and chronic inflammation will be avoided [1]. Furthermore, as an element, Mg is essential to the human body and, as metal, has mechanical properties close to the ones of bone. Special emphasis should be given to children, a sector of the population with a high incidence of bone damage [2]. Bone repair in children is a fast process, and the main requirement from an implant for appropriate healing is to reduce bone load bearing. Therefore, an additional and undesirable immobilisation of the patients will always be necessary to remove a permanent implant. Growing long bones have specific cartilaginous discs at both ends, growth plates, responsible for endochondral ossification and bone formation until adult stage. Any damage due to the implant application or removal, as well as due to the degradation products, could generate irreversible malformations [3,4].

Foetal bone development starts with stem cells condensation, chondrocyte differentiation, proliferation, maturation and ossification. Every step is characterised by changes in cell morphology, proliferation and extracellular matrix (ECM) production, and by a complex molecular regulation [5]. After stem cell condensation, most of the cells become chondrocytes with a rounded morphology and express specific genes such as SRY (sex determining region Y)-box9 (SOX9), aggrecan (ACAN; a proteoglycan) and collagen, type II COL2. The ECM is rich in COL2 and glycosaminoglycans (GAG). Then chondrocytes proliferate and synthesize more ECM, enlarging cartilage [[6], [7], [8], [9]]. Chondrocytes undergo hypertrophy (or maturation), showing a notably enlarged size, a high expression of collagen, type-X gene (COL10) [10,11], and regulating mineralisation of surrounding matrix by expressing other gene markers which are also bone markers, such as osteopontin (OPN) and collagen, type I alpha 1 (COL1A1) [5]. At the final stage of maturation, the chondrocytes can undergo apoptosis.

Previous proteomic studies of mesenchymal stem cell (MSC) chondrogenesis [[12], [13], [14], [15], [16], [17]] have shown that the majority of regulated proteins are related to cell metabolism (e.g., adenosine triphosphate (ATP) synthase subunits alpha (α) and beta (β), carbonyl reductase, aldose reductase, α-enolase, dihydropyrimidinase-like 2, glyceraldehyde-3-phosphate dehydrogenase, and glycogen phosphorylase), ECM and cytoskeleton (e.g., annexins, actin-related proteins, biglycan, chondroadherin, collagen α-2(VI) chain, collagen α-3(VI) chain, fibronectin, vimentin, gelsolin, procollagen-lysine, and transforming growth factor-beta-induced protein ig-h3 precursor), and response to stress (e.g., 78 kDa glucose-regulated protein, endoplasmin, peroxiredoxin-6, peptidyl-prolyl cis-trans isomerise A, superoxide dismutase, heat shock protein beta-1, and stress-induced phosphoprotein 1). Human umbilical cord perivascular (HUCPV) cells are mesenchymal stem cells (MSC), isolated from the vessels surface of umbilical cords, with a high proliferation rate and strong potential for differentiation into the skeletal lineages (both bone and cartilage) [[18], [19], [20]].

In order to prove the potential of Mg-based materials for application in cartlage treatment, a better understanding of the chondrogenic mechanisms influenced by Mg-based materials degradation is still necessary. This study aimed at (I) evaluating the differently expressed proteins during chondrogenesis under the influence of Mg-materials degradation products, contained in the degradation medium (extracts), and (II) determining which of those proteins are directly involved in the chondrogenic differentiation. For this purpose, differential proteomics via label-free quantification of HUCPV cells driven toward chondrogenesis under the influence of three materials (Pure-Mg, Mg-2Ag and Mg-10Gd) was performed. Silver (Ag) was selected as alloying elements due to its antibacterial properties and gadolinium (Gd) to improve material mechanical properties. Cyto- and biocompatibility of these two alloys have been earlier tested and demonstrated ( [21,22,23,24] for Ag and Gd, respectively).

2. Experimental procedures

2.1. Extract preparation and characterization

Extracts of Pure Mg (Pure-Mg, 99.95%), Mg with 2 wt% silver (Mg-2Ag) and Mg with 10 wt% gadolinium (Mg-10Gd) materials were prepared according to EN ISO standards 10993:5 [25] and 10993:12 [26]. Pure elutes were characterised (composition and pH) and diluted in differentiation medium to obtain a common concentration of Mg (i.e., 6.08 mM).

2.2. Induction of micropellets formation and chondrogenic differentiation

Ethical approval for the isolation of HUCPV was obtained from the Ethik-Kommission der Ärztekammer Hamburg. Umbilical cord samples were provided by Asklepios Klinik Altona immediately after caesarean sections of consenting donors. Cell micromasses were obtained from HUCPV cell (passage 2) pellets after 3 days. Chondrogenesis was then chemically induced for up to 11 days with or without Mg-extracts, followed by proteome analysis. For each group (i.e., control, Pure-Mg, Mg-2Ag, and Mg-10Gd) 3 biological replicates were established. Furthermore, 3 technical replicates (i.e, LC MSMS injections) were performed. Control group refers to micropellets driven toward chondrogenesis without any Mg extract (only differentiation medium). More details can be found in Supplemental experimental procedures.

2.3. Proteomic analysis

Proteins from the pellets were extracted using TissueLyzer II (QIAGEN), tryptic in-solution digested and then desalted.

For liquid chromatography–mass spectrometry (LC-MSMS) measurements, all the tryptic digested peptides were subjected to a nano-flow UPLC-column (DionexUltiMate 3000 RSLCnano, Thermo Scientific, Bremen, Germany) coupled via electrospray ionization (ESI) to an Orbitrap mass spectrometer (Orbitrap-Fusion, Thermo Fisher Scientific).

To compare the relative protein abundance, raw data files obtained from the LC-MSMS were processed by MaxQuant 1.5.2.8 [27]. These parameters were used for identification and label-free quantification: identification of the peptides against SwissProt database downloaded from UniProt in July 2015 (with internal contaminants database of MaxQuant); trypsin was used as an enzyme with one missed cleavage; carbamidomethylation on cysteine was set as fixed modification and oxidation of methionine as variable modifications; precursor mass of 20 ppm and fragment mass tolerance of 0.5 Da; and minimum peptide length of 6 amino acids for identification and match between runs.

Peptide spectrum match (PSM) and protein false discovery rate (FDR) were 0.01; and at least 2 ratio count for LFQ was used.

Perseus 1.5.2.6 [28] and Wolfram Mathematica 10.0 (Wolfram Research Europe Ltd., Oxfordshire, United Kingdom) were used for bioinformatics analysis.

Heat maps (Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5; Fig. A1), based on two-sided student's T test, prepared in Perseus, indicates the fold change and significance of each protein of HUCPV cells incubated for 11 days with Mg-alloys (Mg-10Gd, Mg-2Ag, and Pure-Mg) compared to control cells after 11 days incubation without Mg-alloys (permutation-based FDR of 0.01, s0 = 0.1).

Fig. 1.

The number of regulated proteins with more than two-fold change in at least one of the Mg-alloys sorted according to (a) their location in the cells and (b) their involvement in physiological processes.

Fig. 2.

Heat-map and hierarchical clustering of the up- and down-regulated proteins involved in chondrogenesis and cartilage development (P-value = 0.05; min. fold-change of 2) in all Mg-alloys compared based on the mean values of the biological replicates (normalized to Control).

Fig. 3.

Heat-map and hierarchical clustering of the up- and down-regulated proteins involved in apoptosis (P-value = 0.05; min. fold-change of 2) in all Mg-alloys compared based on the mean values of the biological replicates (normalized to Control).

Fig. 4.

Heat-map and hierarchical clustering of the up- and down-regulated proteins involved in cellular response to toxicity (P-value = 0.05; min. fold-change of 2) in all Mg-alloys compared based on the mean values of the biological replicates (normalized to Control).

Fig. 5.

Heat-map and hierarchical clustering of the up- and down-regulated proteins involved in angiogenesis and bone formation (P-value = 0.05; min. fold-change of 2) in all Mg-alloys compared based on the mean values of the biological replicates (normalized to Control).

Other and more detailed experimental procedures are described in Supplemental experimental procedures.

3. Results

3.1. Composition of the extracts

As it can be observed in Table 1, Mg contents increased strongly in the extracts compared to the extraction media (α-MEM supplemented with 10% foetal bovine serum for mesenchymal stem cells (SC-FBS; Stem Cell Technologies, Vancouver, Canada) and 1% antibiotics Penicillin/Streptomycin (Pen strep; Invitrogen, Bremen, Germany)) while Ca and P ones decreased. To avoid osmotic choc and in order to study the effect of alloying element independently of Mg content, extracts were diluted with differentiation medium to obtain a common Mg concentration of about 6.08 mM.

Table 1.

Elemental characterisation of the extraction medium (growth medium) initial extracts (pure) and after dilution to a Mg concentration of 6.08 mM (diluted) measured via ICP-MS. All concentrations are in millimolar (mM).

			Mg (mM)	Ca (mM)	P (mM)	Gd (mM)	Ag (mM)	pH
Extract	Pure	Pure-Mg	51.43	0.70	0.30	n.d.	n.d.	8.68
		Mg-10Gd	80.64	0.32	0.32	2.16 x 10−3	n.d.	8.51
		Mg-2Ag	50.60	0.54	0.54	n.d.	71 x 10−3	8.68
	Diluted	Pure-Mg	6.08	1.79	1.26	n.d.	n.d.	8.15
		Mg-10Gd	6.08	1.77	1.26	1.63 x 10−4	n.d.	8.15
		Mg-2Ag	6.08	1.72	1.16	n.d.	11 x 10−3	8.25
Growth or expansion medium			0.81	1.80	1.01	n.d.	n.d.	7.34
Differentiation or chondrogenic medium			0.82	1.89	1.37	n.d.	n.d.	7.40

3.2. Effects of the Mg-alloys degradation products on chondrogenic-differentiated HUCPV proteome

In order to determine the influence of Pure-Mg, Mg-10Gd, and Mg-2Ag extracts on HUCPV cells driven toward chondrogenesis, proteins from each condition were analysed with differential bottom-up proteomics using label-free quantification (LFQ).

246 significantly regulated proteins (Table A1) were found under the influence of the extracts and clustered in a heat map (Fig. A1). 136 proteins were upregulated in the presence of Mg-10Gd, Mg-2Ag and Pure-Mg (from which 134 proteins were common for the three extracts) while 110 proteins were downregulated. A Gene Ontology (GO) annotation downloaded from UniProt was performed for each protein of the list of regulated proteins. Clustering these proteins regarding their localisation in the cells (cellular compartment; Fig. 1a) indicated that the number of upregulated extracellular proteins and membrane proteins (involved not only in ECM composition) was considerably higher than downregulated ones in the presence of Mg alloys. Additionally, the number of regulated cytosol proteins was high. Cytosolic and cytoskeletal proteins were mostly downregulated. Fig. 1b shows the most affected biological processes. The highest number of regulated proteins were involved in cell binding, differentiation, apoptosis, and cell proliferation. To a lesser extent, proteins involved in angiogenesis, energy metabolism, bone development, and chondrogenesis were influenced by the extracts. Proteins involved in chondrogenesis and cartilage formation are depicted in Fig. 2. Heat maps in Fig. 3, Fig. 4, Fig. 5 illustrate the regulated proteins clustered according to their involvement in apoptosis, response to cell toxicity and angiogenesis, respectively.

In a second step, Mg-alloy specific proteins will be discussed.

3.2.1. Regulated proteins involved in chondrogenesis and cartilage formation

Fig. 2 illustrates the regulated chondrogenesis-related proteins. Four chondrogenesis-related proteins were upregulated by the extracts: glycosylphosphatidylinositol specific phospholipase D1 (GPLD1) was upregulated in the presence of Mg-alloys, while hexosaminidase B (beta polypeptide) (HEXB) and transforming growth factor, beta-induced, 68 kDa (TGFBI) were upregulated compared to the control. In addition, proteins involved in cartilage ECM formation and organisation (fibronectin 1 (FN1), collagen, type VI (COL6), tenascin C (TNC), intercellular adhesion molecule 1 (ICAM1), vitronectin (VTN), heparan sulfate proteoglycan 2 (HSPG2), procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 and 2 (PLOD1 (significantly in the presence of pure-Mg and Mg10Gd) and PLOD2) and integrins α-2 (ITGA2), α-5 (ITGA5)) were upregulated in the presence of Mg. COL1A1 was downregulated by all the extracts. ITGA5 was significantly upregulated in the presence of Mg-10Gd but not with the other extracts.

3.2.2. Regulated proteins involved in apoptosis

Regulated proteins involved in apoptosis were clustered in the heat map shown in Fig. 3. From the total 49 proteins identified, 29 were upregulated, while 20 of them were downregulated in the presence of Mg-alloys. S100 calcium binding protein A9 (S100A9) was completely absent in the presence of Mg-alloys. Galectin 3 (LGALS3) was downregulated in the presence of Mg-2Ag (in less than 2 fold) and absent in any biological replicates in the presence of the other Mg-alloys. On the other hand, 10 apoptotic-related regulated proteins in at least two biological replicates of each condition were only present after incubation of HUCPV with Mg-alloys. From those proteins, 6 were stimulators of apoptosis: C3, Kininogen-1 (KNG1), prostaglandin-endoperoxide synthase 2 (PTGS2), TAR DNA-binding protein (TARDBP), SERPINA3 and GPLD1 while 4 were inhibitors: apolipoprotein E (APOE), ICAM1, niban-like protein 1 (FAM129B) and angiopoietin-related protein 4 (ANGPTL4). Regarding the downregulated proteins in the presence of the extracts, programmed cell death protein 5 (PDCD5) and PRKC apoptosis WT1 regulator protein (PAWR) were positive regulators of apoptosis. The regulation of the other apoptotic-related proteins is significant in all Mg-alloys.

3.2.3. Regulated proteins involved in the cellular response to toxicity

The heat map in Fig. 4 indicates the regulated proteins involved in cellular response to toxic substances in the presence of Mg-alloys. 6 of those 10 proteins involved in the cellular response to toxicity were upregulated while 4 were downregulated. ICAM1, C3, and paraoxonase 1 (PON1) were only present in the HUCPV cells incubated with Mg-alloys.

3.2.4. Regulated proteins involved in angiogenesis

18 of angiogenesis-related proteins were regulated in the presence of Mg-alloys, 17 of those were significantly increased in the presence of at least one of the Mg-alloys. Apolipoprotein D (APOD), Complement Component 3 (C3), PTGS2, GPLD1, Alpha-1-antichymotrypsin (SERPINA3) and angiopoietin-like 4 (ANGPTL4) were present in at least two biological replicates of the HUCPV cells incubated with Mg-alloys, and not present in the control (Fig. 5). Ribonuclease/angiogenin inhibitor 1 (RNH1) was downregulated in the presence of all Mg-alloys. However, downregulation of this protein is significant only in the presence of Mg-2Ag. Moreover, ITGA5 was significantly upregulated in the presence of Mg-10Gd, while there was no significant change in the presence of the other Mg-alloys. Thrombospondin 1 (THBS1) was upregulated with all the extracts. The upregulation of the other angiogenesis-related proteins was significant in all Mg-alloys.

3.2.5. Regulated proteins in all biological replicates in the presence of Mg-10Gd & Mg-2Ag

The significantly regulated proteins in the presence of Mg-10Gd and Mg-2Ag (5 proteins) are listed in Table 2. Charged multivesicular body protein 4B (CHMP4B) was upregulated (while downregulated with Pure-Mg) and Asparagine synthetase (NARS) was downregulated by the three extracts (being also significantly decreased with Mg-10Gd and Mg-2Ag compared to Pure-Mg. Both proteins are involved in cell apoptosis and NARS also in response to toxic substrates. Thioredoxin reductase 1 (TXNRD1) was downregulated only with Mg-2Ag and Mg-10Gd. Keratin 19 (KRT19) was downregulated with the three extracts (but the downregulation was significantly lower than with Pure-Mg (Table A1). SERPINE2, an ECM protein, was present only in the incubated cells with the three Mg-alloys, exhibiting higher expression in Mg-10Gd and Mg-2Ag (in all of the biological replicates of Mg-10Gd and Mg-2Ag) than in Pure-Mg (Table A1).

Table 2.

Significantly regulated proteins (⇧ upregulation - ⇩ downregulation) under different conditions. Function based on UniProt database search. Protein names and symbol are according to Hugo Gene Nomenclature Committee – synonyms/previous names are italicised.

	Protein name (gene name) synonym	Function	Fold change in Pure-Mg	Fold change in Mg-10Gd	Fold change in Mg-2Ag
Significantly regulated proteins in Mg-10Gd and Mg-2Ag (not in Pure-Mg).	Charged multivesicular body protein 4B (CHMP4B) Chromatin modifying protein 4B	negative regulation of cell death	/	⇧ 2.29 ±0.199	⇧ 2.57 ±0.068
	Eukaryotic translation initiation factor 3 subunit C (EIF3C)	translation initiation factor activity	/	⇩ 2.04 ±0.0.97	⇩ 2.34 ±0.056
	Thioredoxin reductase 1 (TXNRD1)	cell proliferation, response to reactive oxygen species, thioredoxin-disulfide reductase activity	/	⇩ 2.14 ±0.02	⇩ 2.24 ±0.018
	Keratin 19 (KRT19)	cell differentiation, involved in embryonic placenta development, structural constituent of cytoskeleton	/	⇩ 16.22 ±0.319	⇩17.78 ±0.068
	Asparagine-tRNA ligase (NARS)	negative regulation of apoptotic process, response to toxic substance	/	⇩ 4.17 ±0.083	⇩ 5.13 ±0.069
			Fold change in Pure-Mg	Fold change in Mg-10Gd	Fold change in Mg-2Ag
Significantly regulated proteins in Mg-10Gd and Pure-Mg (not in Mg-2Ag)	Transmembrane p24 trafficking protein 7 (TMED7) Transmembrane emp24 domain-containing protein 7	protein transport	⇧ 3.39 ±0.077	⇧ 2.82±0.0056	/
	Sideroflexin 3 (SFXN3)	transporter activity	Not present in control	Not present in control	/
	IKBKB interacting protein (IKBIP) Inhibitor of nuclear factor kappa-B kinase-interacting protein	response to X-ray	⇧ 2.09 ±0.036	⇧ 2.29 ±0.072	/
	Pyrophosphatase (inorganic) 1 (PPA1) PP	magnesium ion binding; phosphate-containing compound metabolic process	⇩ 2.45 ±0.022	⇩ 2.00 ±0.060	/
	Bleomycin hydrolase (BLMH)	aminopeptidase activity, proteolysis, response to toxic substance	Not present in Pure-Mg	⇩ 2.19 ±0.056	/
	Four and a half LIM domains protein 1 (FHL1) LIM protein SLIMMER (SLIM1)	cell differentiation, positive regulation of potassium ion transport, zinc ion binding	⇩ 3.31 ±0.194	⇩ 2.69 ±0.065	/
	Nexilin F-actin binding protein (NEXN) NELIN, nexilin	regulation of cytoskeleton organisation	Not present in Pure-Mg	Not present in Mg-10Gd	/
	AHNAK nucleoprotein (AHNAK) neuroblast differentiation-associated protein, desmoyokin	regulation of voltage-gated calcium channel activity	⇩ 2.57 ±0.098	⇩ 2.69 ±0.24	/
	Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (PLOD1) lysyl hydroxlase 1, LH1	oxidation-reduction process, procollagen-lysine 5-dioxygenase activity	⇧ 2.09 ±0.020	⇧ 2.00 ±0.133	/
	tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein epsilon (YWHAE) 14-3-3 protein epsilon	negative regulation of cysteine-type endopeptidase activity involved in apoptotic process, positive regulation of protein insertion into mitochondrial membrane, involved in apoptotic signalling pathway	⇩ 2.09 ±0.117	⇩ 2.24 ±0.178	/
	Prostaglandin-endoperoxide synthase 2 (PTGS2) prostaglandin G/H synthase 2, cyclooxygenase 2, COX2	positive regulation of apoptotic process, angiogenesis, involved in sprouting angiogenesis, bone mineralisation	Not present in control	Not present in control	/
	tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ) protein kinase C inhibitor protein 1	positive regulation of protein insertion into mitochondrial membrane involved in apoptotic signalling pathway	⇩ 2.24 ±0.067	⇩ 2.75 ±0.078	/
	ATP synthase, H+ transporting, mitochondrial Fo complex subunit B1(ATP5F1)	ATP biosynthetic process, transporter activity	⇧ 2.19 ±0.061	⇧ 2.63 ±0.132	/
	Galectin 3 (LGALS3) lectin, galactoside-binding, soluble, 3	epithelial cell differentiation, regulation of extrinsic apoptotic signalling pathway via death domain receptors, regulation of T cell apoptotic process, regulation of T cell proliferation	Not present in Pure-Mg	Not present in Mg-10Gd	/
	Plastin 3 (PLS3) T-plastin	auditory receptor cell differentiation, bone development, calcium ion binding	⇩ 2.04 ±0.071	⇩ 2.09 ±0.068	/
	Cytochrome c oxidase subunit 4 isoform 1 (COX4I1) cytochrome c oxidase subunit IV, COX4	generation of precursor metabolites and energy, response to nutrient	⇧ 2.19 ±0.066	⇧ 2.04 ±0.041	/
	Calpain 1 (CAPN1) calpain 1, (mu/I) large subunit	extracellular matrix disassembly, positive regulation of cell proliferation, proteolysis	⇩ 3.47 ±0.083	⇩ 3.16 ±0.061	/
	Lecithin-cholesterol acyltransferase (LCAT) phosphatidylcholine-sterol acyltransferase	lipoprotein biosynthetic process, response to copper ion	Not present in control	Not present in control	/
	H2A histone family, member (H2AFY) Core histone macro-H2A.1, MACROH2A1	SH3/SH2 adaptor activity	⇧ 2.04 ±0.099	⇧ 2.29 ±0.108	/
	Actinin alpha 4 (ACTN4)	BAT3 complex binding, positive regulation of ER-associated ubiquitin-dependent protein catabolic process	⇩ 2.09 ±0.035	⇩ 2.19 ±0.027	/
	Orosomucoid 1 (ORM1) alpha-1-acid glycoprotein 1, OMD, ORM	metal ion binding, SMAD protein signal transduction, transport	Not present in Pure-Mg	Not present in Mg-10Gd	/
	Alpha fetoprotein (HPAFP)	oxygen transporter activity, heme binding	⇩ 2.51 ±0.0137	⇩ 2.88 ±0.202	/
			Fold change in Pure-Mg	Fold change in Mg-10Gd	Fold change in Mg-2Ag
Significantly regulated proteins in Mg-2Ag and Pure-Mg (not in Mg-10Gd)	REX2, RNA exonuclease 2 homolog (S. cerevisiae) (REXO2), Oligoribonuclease, mitochondrial precursor	3′-5′ exonuclease activity, focal adhesion, nucleotide metabolic process	Not present in control	/	Not present in control
	LIM domain and actin-binding protein 1 (LIMA1), Epithelial protein lost in neoplasm, EPLIN, FLJ38853	focal adhesion, negative regulation of actin filament depolymerisation, stress fiber	⇩ 4.07 ±0.036	/	⇩ 2.51 ±0.112
	Reticulon-4 (RTN4), ASY, Foocen, KIAA0886, My043, Nbla00271	negative regulation of cell growth, of apoptotic process, regulation of cell migration	⇩ 2.19 ±0.160	/	⇩ 2.14 ±0.14
	Histone cluster 1, H2bl (HIST1H2BL) Histone H2B type 1-L, H2BFC, Histone H2B.c	nucleosome assembly	⇧ 2.24 ±0.044	/	⇧ 2.75 ±0.035
	Kinectin (KTN1), CG1, CG-1 antigen, kinesin receptor	microtubule-based movemen	Not present in Pure-Mg	/	Not present in Mg2Ag
	non-POU domain containing, octamer-binding (NONO), NonO protein, Non-POU domain-containing octamer-binding protein	DNA recombination, DNA repair, negative regulation of oxidative stress-induced neuron intrinsic apoptotic signaling pathway [	⇧ 2.45 ±0.097	/	⇧ 2.24 ±0.040
	LIM and SH3 domain protein 1 (LASP1), Metastatic lymph node gene 50 protein,Lasp-1	focal adhesion;ion transmembrane transporter activity, ion transport, zinc ion binding	⇩ 4.07 ±0.089	/	⇩ 2.88 ±0.23
	Major vault protein (MVP), LRP, Lung resistance-related protein, Major vault protein	ERBB signaling pathway, protein transport	⇧ 2.29 ±0.115	/	⇧ 2.04 ±0.006
	Caldesmon 1 (CALD1), CDM, L-caldesmon, Non-muscle caldesmon	movement of cell or subcellular component, muscle contraction	⇩ 3.09 ±0.076	/	⇩ 2.04 ±0.090
	Nucleobindin 1 (NUCB1), CALNUC, DKFZp686A15286	regulation of protein targeting	⇧ 2.34 ±0.108	/	⇧ 2.40 ±0.008
	heat shock 10 kDa protein 1 (chaperonin 10), (HSPE1), 10 kDa heat shock protein, 10 kDa chaperonin, CPN10	activation of cysteine-type endopeptidase activity involved in apoptotic process, osteoblast differentiation	⇧ 2.24 ±0.107	/	⇧ 2.24 ±0.029
	Branched-chain-amino-acid aminotransferase 1, cytosolic (BCAT1), BCAT(c), BCATC, BCT1, Branched-chain-amino-acid aminotransferase, cytosolic	aspartate biosynthetic process, cell proliferation	⇩ 2.24 ±0.034	/	⇩ 2.14 ±0.103
	Tu translation elongation factor, mitochondrial (TUFM), Elongation factor Tu, mitochondrial, P43, COXPD4	GTP binding, GTPase activity, mitochondrial translational elongation [	⇩ 2.29 ±0.041	/	⇩ 2.29 ±0.105
	Serum amyloid A4, constitutive (SAA4), Serum amyloid A-4 protein, CSAA, C-SAA	acute-phase response, cell chemotaxis, chemoattractant activity	Not present in control	/	Not present in control
	Phosphatidylethanolamine-binding protein 1 (PEBP1), HCNP, HCNPpp, Neuropolypeptide h3, PBP	ATP binding, serine-type endopeptidase inhibitor activity	⇩ 3.89 ±0.110	/	⇩ 2.88 ±0.117
	Filamin-A, akoga (FLNA), ABP-280, ABPX, Actin-binding protein 280, Alpha-filamin	cell junction assembly, focal adhesion, negative regulation of apoptotic process, wound healing	⇩ 2.14 ±0.062	/	⇩ 2.09 ±0.020
	Spermidine synthase (SRM), PAPT, Putrescine aminopropyltransferase, SPDSY	polyamine metabolic process, protein homodimerization activity, spermidine biosynthetic process	⇩ 2.29 ±0.152	/	⇩ 2.40 ±0.067
	Ezrin (EZR), CVIL, CVL, Cytovillin, DKFZp762H157, Ezrin	focal adhesion, regulation of cell shape	⇩2.19 ±0.116	/	⇩ 2.29 ±0.017
	Heat shock 60 kDa protein 1 (chaperonin) (HSP60), 60 kDa heat shock protein,Chaperonin 60, Chaperonin 60, CPN60, GROE	negative regulation of apoptotic process, positive regulation of interleukin-6,10,12	⇧ 2.14 ±0.099	/	⇧ 2.00 ±0.045
	l-lactate dehydrogenase B (LDHB), l-lactate dehydrogenase B chain, LHD heart subunit, LHD-B	l-lactate dehydrogenase activity, pyruvate metabolic process	⇩ 2.04 ±0.034	/	⇩ 2.09 ±0.049
	Apolipoprotein A-II (APOA2), apoAII, ApoA-II, Apo-AII, Apolipoprotein A2	acute inflammatory response, protein oxidation	Not present in control	/	Not present in control
	Crystallin alpha B (CRYAB), Alpha-crystallin B chain, Alpha (B)-crystalline, HspB5, HSPB5	negative regulation of extrinsic apoptotic signaling pathway, positive regulation of cell aging, positive regulation of osteoblast differentiation, negative regulation of adipose tissue development,	⇧ 3.02 ±0.084	/	⇧ 2.45 ±0.048
	Serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1 (SERPINA1), A1A, A1AT, Alpha-1-antiproteinase, alpha-1-antitrypsin	acute-phase response, inflammatory response, serine-type endopeptidase inhibitor activity	Not present in control	/	Not present in control
	Serpin peptidase inhibitor, clade C (antithrombin), member 1 (SERPINC1) Antithrombin-III, AT3, ATIII, Serpin C1	acute-phase response, serine-type endopeptidase inhibitor activity	Not present in control	/	Not present in control
	Haptoglobin-related protein (HPR), A-259H10.2, Haptoglobin-related protein, HP	extracellular matrix disassembly, negative regulation of cell proliferation, negative regulation of cell-cell adhesion mediated by cadherin, positive regulation of fibrinolysis, serine-type endopeptidase activity	Not present in control	/	Not present in control
	Glutamic-oxaloacetic transaminase 2, mitochondrial (aspartate aminotransferase 2) (GOT2), Aspartate aminotransferase, mitochondrial, FABP-1, FABPpm	canonical glycolysis,epithelial cell differentiation	⇧ 2.63 ±0.088	/	⇧ 2.51 ±0.48
	Glutaredoxin 3 (GLRX3), Glutaredoxin-3, GRX3, GRX4, PICOT, PKC-interacting cousin of thioredoxin	osteoblast differentiation, regulation of apoptotic process	Not present in P-Mg	/	Not present in Mg-2Ag
	Programmed cell death protein 5 (PDCD5), Protein TFAR19, TF-1 cell apoptosis-related protein 19, TFAR19	chloride transmembrane transport, lipid transport	⇩ 3.63 ±0.067	/	⇩ 2.63 ±0.196
	Importin5 (IPO5), Imp5, Importin-5, Importin subunit beta-3	positive regulation of apoptotic process, positive regulation of intrinsic apoptotic signaling pathway	⇩2.14 ±0.039	/	⇩ 2.19 ±0.008

3.2.6. Regulated proteins in the presence of Mg-10Gd & Pure-Mg

Regulated proteins (22 proteins) in all of the biological replicates in HUCPV cells incubated with Mg-10Gd and Pure-Mg are listed in Table 2. Among them, 6 and 9 proteins expression was increased and decreased, respectively, compared to control cells. Three proteins were identified only in the presence of Mg-10Gd and Pure-Mg, whereas 3 proteins were absent in the presence of both extracts. Four proteins involved in the apoptotic process were regulated: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptide (YWHAE) and LGALS3 were downregulated, the latter being absent in cells incubated with those extracts. PTGS2 was remarkably upregulated with the three extracts, but significantly higher with Mg-10Gd and Pure-Mg than with Mg-2Ag. Among the proteins involved in transport, sideroflexin 3 (SFXN3) involved in iron homeostasis was absent in control cells and orosomucoid 2 (ORM2) was missing in the presence of Mg-alloys. Four downregulated proteins with a role in cell differentiation, four and a half LIM domains protein 1 (FHL1), PLS3, LGALS3, and calpain 1 (CAPN1) are listed in Table 2. Additionally, downregulation of calmodulin 1 (CALM1) was observed with the three extracts (although not significantly), being more notable with Mg-10Gd and Mg-Pure than with Mg-2Ag.

3.2.7. Regulated proteins in the presence of Mg-2Ag & Pure-Mg

In the presence of Mg-2Ag and pure-Mg, 29 proteins were significantly regulated in all of the biological replicates of these two conditions (Table 2). Eight of these proteins were up- and 13 of them were down-regulated. Six proteins were only present in all biological replicates of the incubated cells with Mg-2Ag and pure-Mg. Furthermore, there are two proteins which were presented only in the cells without Mg-alloys. Regulated proteins involved in apoptosis: Reticulon 4 (RTN4), Filamin-A (FLNA), Glutaredoxin-3 (GLRX3) and Importin-5 (IPO5) were down-regulated in the presence of Mg-2Ag and pure-Mg. 10 kDa heat shock protein (HSPE1) was up-regulated while 60 kDa heat shock protein (HSPE1) and Alpha-crystallin B chain (CRYAB) were up-regulated. Regarding proteins involved in cell differentiation, Aspartate aminotransferase (GOT2) was up-regulated and Glutaredoxin-3 (GLRX3) was down-regulated in cells cultured with Mg-2Ag and pure Mg. Moreover, the proteins involved in transportation such as LIM and SH3 domain protein 1 (LASP1), Major vault protein (MVP), Ezrin (EZR), Programmed cell death protein 5 (PDCD5), were down- or up-regulated in the presence of Mg-2Ag and pure-Mg. Among the proteins observed in the presence of Mg-2Ag and pure-Mg, which were absent in control cells, APOA2, Antithrombin-III (SERPINA3), and Alpha-1-antitrypsin (SERPINA1) are involved in the acute-phase response. Non-POU domain-containing octamer-binding protein (NONO) was upregulated in the presence of Mg-2Ag and pure-Mg.

4. Discussion

According to the overall results, it is obvious that the increased concentration of Mg²⁺ ions is responsible for the main effects observed in this study. In comparison to the effect of Mg²⁺ ions, Ag⁺-ions and Gd³⁺-ions have minor effects. Additionally, increased extracts pH probably have an influence on chondrogenesis. Indeed, lower pH (as observed in diabetes and aging) negatively influence bone homeostasis (altered bone structure and density). Furthermore, an alkaline pH (about 8) is optimal for alkaline phosphatase activity and hydroxyapatite precipitation while switching off osteoclast resorption [29,30]. Moreover, Moghadam et al. demonstrated that chondrogenesis was more efficient after short-term culture in alkaline medium [31]. In vitro and even in vivo magnesium-based material degradation is a complex mechanism accompanied by increased pH, ion released (increased osmolality) and other phenomenon. Therefore, the already observed positive effects of these biomaterials on bone healing are probably multifactorial and due to the synergistic effects of magnesium-based degradation. Furthermore, pH of the different extracts are similar thus, the proteomics variation measured between the different extracts are probably due to the material compositions themselves.

Mg²⁺ is an endogenous element in living organisms and its doubly charged ion involved in a multitude of physiological processes, in many cases enabling defined functions of proteins as their ligands. Living organisms are equipped with a fine-tuned system guaranteeing constant levels of Mg ions in the intra- and extracellular space. Thus, it is not surprising that the increase of Mg ions in the culture medium, will lead to an active cell reaction (e.g., regulation of 246 proteins). Extracellular proteins and cytosolic/cytoskeletal proteins were mostly upregulated and downregulated, respectively. Among the main cell functions of the proteins influenced by the extracts, cell attachment, growth, differentiation and survival or apoptosis were identified. Such functions are involved also in the interactions between chondrocytes and ECM and are important for cartilage homeostasis and cartilage repair [32]. Mg has a key role in cellular energy metabolism and Mg²⁺ ions are known to enhance the activity of adenosine triphosphate (ATP) synthase [33]. This enzyme consists of two main regions, F₀ and F₁ themselves composed of subunits. Here, accordingly, several subunits were upregulated (Fig. A1): from the F₀ complex: ATP synthase, H⁺ transporting, mitochondrial F0 complex, subunit B1 (ATP5F1) and from F₁: ATP synthase, H⁺ transporting, mitochondrial F1 complex, alpha subunit 1 (ATP5A1), gamma polypeptide 1 (ATP5C1), beta polypeptide (ATP5B) and O subunit (ATP5O). Similarly, upregulation of voltage-dependent anion channel 1 (VDAC1) was induced by the extracts. This protein interacts with hexokinase and creatine kinase to convert newly generated ATP into high-energy storage molecules. Therefore the increased synthesis of VDAC1 is also associated with high metabolically active and energy-demanding cells [34]. A possible explanation may be that the increased Mg²⁺-concentration induces the increased synthesis of energy-rich (phosphate-rich) metabolites for binding free Mg²⁺- ions for maintaining Mg²⁺ homeostasis. Increased of energy-rich metabolites may increase biosynthesis by which the energy-rich metabolites are consumed [35].

Proteins involved in cholesterol metabolism were strongly upregulated by the extracts. Lecithin-cholesterol acyltransferase (LCAT) showed 2-fold higher expression with Mg-10Gd and Pure-Mg than with Mg-2Ag. This protein is the central enzyme involved in the extracellular metabolism of lipoproteins. Apolipoprotein A-I (APOA1) is the most potent phosphatidylcholine-sterol acyltransferase activator in plasma (although it can also be activated by APOE, APOC1 and APOA4). All those apolipoproteins involved in cholesterol efflux (as well as additional ones as APOD) were strongly upregulated with the three extracts (Appendix A). Both LCAT and APOAI lack or deficiency give rise to cartilage degeneration and the development of osteoarthritis (OA) [36,37]. Thus, the possible inhibitory effect of those extracts on OA (i.e., through the upregulation of the aforementioned proteins), is an interesting subject for future investigations.

The three extracts induced the upregulation of proteins involved in cartilage development (both ECM integrity and ECM-cell adhesion) (Appendix A). Among them, TNC is notably upregulated during cartilage development, and it is involved in ECM remodelling and cell differentiation [38]. HSPG2 is involved in the metabolism (synthesis and catabolism) of GAG, one of the main components of cartilage ECM. It is required for cartilage development, where it plays a role in ECM organization. ICAM1 (whose expression was not detected in control cells) has multiple functions, being relevant its role in cell adhesion (specifically integrin-mediated adhesion). Its expression in human chondrocytes can be induced by exogenous interleukin 1 α (IL1α), which was added to the culture medium in the study of Davies et al. in order to induce chondrogenesis [39]. Those results suggest a synergistic effect of IL1α in the presence of Mg-extracts or a direct effect of the Mg ions on ICAM1 expression.

Another group of proteins, upregulated by the three extracts, was the integrin family. Integrins are responsible for primary adhesion of cells to orthopaedic or dental implants, therefore addition of Mg ions to the surface of biomaterials enhance cell-material interaction, reducing the possibilities of implant rejection by the body [40]. Furthermore, the integrin family plays a major role in mediating cell-matrix interactions that are important in regulating cartilage development and repair. Integrins and cell-matrix interactions have been shown to be involved in chondrogenesis of MSC [38] and enhance MSC attachment to endochondral defects (enhancing its repair). Additionally, integrins are involved in the negative regulation of apoptosis. Upregulation of integrins in response to Mg extracts seems therefore beneficial, not only for enhancing chondrogenesis of HUCPV cells, but also for generating a good quality cartilaginous matrix. In native cartilage, chondrocytes express several members of the integrin family, which can serve as receptors for relevant proteins in the structure of the ECM (which also were upregulated under the influence of the extracts): ITGA5 is a receptor for FN1, ITGAV for VTN and ITGA2 for COL6. Since divalent cations, including Mg²⁺, are ligands for integrins and activate them, an increased cell adhesion of HUCPV cells under the influence of the three extracts is expected. The integrin-signalling proteins are important components of the cartilage ECM. VTN interacts with glycosaminoglycans and proteoglycans and serves as a cell-to-substrate adhesion molecule. Furthermore, it inhibits the membrane-damaging effect of the terminal cytolytic effect of the complement pathway. FN1 is involved in early chondrocyte differentiation events after birth. Its upregulation takes place during the condensation of stem cells [41]. COL4 is found in connective tissue. A notable upregulation of this protein has been reported during early stages of human MSC chondrogenesis (after 10 days), possibly due to the influence of this protein on Sox9 [42]. Two other chondrogenic-related proteins were upregulated by the three extracts: GPLD1, which stimulates chondrocyte differentiation and HEXB, which has a role in the catabolic process of chondroitin sulphate.

The presence of Mg-extracts on HUCPV cells during chondrogenesis also induced the upregulation of TGFBI, a protein involved in the cell-collagen interaction, and important for ECM remodelling during chondrocyte differentiation [43]. TGFBI overexpression positively enhances the proliferation and chondrogenic potential of human synovium-derived MSC [44]. Furthermore, TGFBI induces upregulation of integrins [45]. Therefore, Mg extracts may induce an enhancement of TGFBI, which will in turn upregulate integrin production, and subsequently, integrin-mediated cell adhesion and chondrogenesis. In addition, TGFBI induces expression of PLOD1 and PLOD2, proteins upregulated in regenerated cartilage in vivo (regarding the natural cartilage).

Angiogenesis is a fundamental component of bone repair due to the development of blood vessels in the fracture callus [46] and a vital part of bone formation [46,47]. Hypertrophic cartilage produces angiogenic stimulators [[48], [49], [50]], unlike angiogenesis inhibitors, which are secreted by immature chondrocytes [51,52]. The 3 processes, chondrogenesis, angiogenesis and bone formation are closely related. Hence, some of the regulated proteins are involved in all of them (e.g., ITGA5 and COL1A1). Upregulation of 15 of the 16 regulated proteins involved in angiogenesis shows a hypertrophic stage of chondrocytes. Furthermore, THBS1 upregulation could be of interest since it has been shown that this protein inhibits vascular endothelial growth factor (VEGF)-induced migration in human microvascular cells [53].

COL1A1 was downregulated under the influence of the three extract. COL1A1 is involved in bone trabecula formation and final stage of cartilage development. Nevertheless, the downregulation of this protein versus the lack of effect on COL2 production is indicating a reduction in the ratio COL2/COL1 characteristic in cartilage tissue. PLS3 is involved in bone development and its downregulation in the presence of Mg-alloys may avoid cartilage mineralisation. Consequently, the downregulation of those two proteins is beneficial in order to keep the chondrogenic phenotype of the differentiated HUCPV cells. Furthermore, two proteins PTGS2 and GPLD1 were only detected in the presence of extracts. PTGS2 (or cyclooxygenase 2, COX 2) is responsible for production of inflammatory prostaglandins. Furthermore, PTGS2 is also associated with increased cell adhesion, phenotypic changes and resistance to apoptosis. PTGS2 is a target of nonsteroidal anti-inflammatory drugs (NSAID) including acetylsalicylic acid (“aspirin”) and isobutylphenylpropionic acid (“ibuprofen”). NSAID have been reported to have (controversial/negative) influence osteogenesis during bone fracture healing [54]. Welting et al. demonstrate the role of PTGS2 in chondrocyte maturation-hypertrophy [55]. GPLD1 hydrolyses the inositol phosphate linkage in proteins anchored by glycosylphosphatidylinositol (GPI) to the outer leaflet of the plasma membrane, thereby releasing the attached protein. Over 250 GPI-proteins are known, among them heparan sulfate proteoglycans (HSPG) [56], ephrin A ligands (for Eph receptors - the largest known subfamily of receptor protein-tyrosine kinases), putative adhesion/signalling molecules of the Ly6 family, and enzymes like alkaline phosphatase [57]. GPI-anchored proteins are believed to have a role in cell adhesion events involved in tissue patterning and cell signalling. Indeed, Ahrens et al. demonstrated that GPI-anchored proteins are necessary to the columnar tissue arrangement and the proper development of the growth plate [57].

Apoptosis is a tightly regulated process, inevitable and essential during development, particularly during formation of articular cartilage and endochondral ossification of growth plate [58]. Increased apoptosis in native cartilage is associated with matrix degradation. Induction of MSC chondrogenesis in vitro using micromasses formation models increases the possibility of apoptosis due to the severe hypoxic conditions that cells suffer in the centre of the spheres. Nevertheless, some differences in protein expression (involved in both positive and negative regulation of apoptosis) were found due to the action of the extracts (Fig. 4). On the one hand, 9 apoptosis-related proteins were found only in presence of extracts: 5 were stimulators of apoptosis (C3, KNG1, PTGS2, TARDBP, and GPLD1) and 4 inhibitors (APOE, ICAM1, FAM129B, and ANGPTL4). On the other hand, 2 proteins stimulating apoptosis were downregulated, S100A9 and LGALS3. S100A9 was completely absent in the presence of Mg-alloys. This protein also has a role in actin cytoskeleton reorganization, proinflammatory response and oxidant-scavenging. LGALS3 was downregulated in the presence of Mg-2Ag, and absent in the presence of Pure-Mg and Mg-10Gd, which may indicate a toxic action of Ag. Galectin-3 (LGALS3) is also found to be a potent inhibitor for osteoclastogenesis in vitro [59]. Those results show a clear influence on apoptosis and suggest a reduction of cell death by the extracts.

Since Mg-alloy degradation products might have undesired side effects like toxicity to the cells, regulated proteins involved in the response to toxic effect were evaluated. Six from the 10 regulated proteins known to be associated with stress response showed increased expression in the presence of extracts. From those six, four proteins were not found in the absence of the extracts, which might suggest a response of the cells toward toxicity; however, these proteins have also other roles in the cells. For instance, C3 is one of the stimulators for angiogenesis and ICAM1 is involved in cell migration and adhesion, therefore reinforcing cartilage repair.

Some proteins were regulated only by 2 extracts, or showed significant differences in the expression (always normalised to control) among the 3 extracts. In principle, proteins up- or downregulated only by Mg-2Ag or Mg-10Gd extracts should give information about the effects of the alloying elements (Ag and Gd) on HUCPV chondrogenesis, as Mg concentration was constant. CHMP4B, having a role apoptosis suppression, was upregulated with those extracts, could be beneficial for cell viability. However, NARS with the same function was downregulated. Regarding the other function of NARS in the cellular response to toxic substrate, its downregulation supports suggests a lack of toxic effect of Mg-2Ag and Mg-10Gd. Interestingly, serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 2 (SERPINE2), was only expressed in the presence of the extracts, and its expression was significantly higher with Mg-10Gd and Mg-2Ag than with Pure-Mg. It has been shown that SERPINE2 expression in human chondrocytes might prevent cartilage catabolism by inhibiting the expression of matrix metallopeptidase 13 (MMP13), one of the most relevant collagenases, involved in cartilage breakdown in OA [60]. Therefore, alloying elements could have a positive effect on the maintenance of cartilage integrity. TXNRD1, protein involved in cell proliferation, was downregulated only with Mg-10Gd and Mg-2Ag. In correlation with this, a strong downregulation of the microtubule-associated protein 9 (MAP9) with Mg-10Gd extract was observed, while a slight upregulation was detected with Pure-Mg and Mg-2Ag. MAP9 is required for mitosis progression and cytokinesis (UniProt). Therefore, its downregulation could decrease cell cycle progression and cell division. The proteins commonly regulated only by Pure-Mg and Mg-2Ag as well as Pure-Mg and Mg-10Gd were mainly involved in apoptosis, indicating that Mg itself has a significant influence on this cellular process.

Mg-2Ag and Pure-Mg showed a beneficial effect on HUCPV viability by downregulating proteins positively involved in apoptosis (RTN4,FLNA, GLRX3, and IPO5) and upregulating two proteins involved in negative regulation of apoptosis (60 kDa heat shock protein and α-crystallin B chain). However, FLNA, having a role in protecting cells from apoptosis was also downregulated. An interesting upregulated protein in the presence of Mg-2Ag and Pure-Mg is non-POU domain containing, octamer-binding protein (NONO). This protein has a protective role in the regulation of oxidative stress-induced neuron intrinsic apoptotic signalling pathway, Furthermore, NONO promotes chondrogenesis by interacting with SOX9 thus allowing/promoting transcription of SOX9 target genes such COL2A1 [61]. Both functions suggest that NONO upregulation is beneficial for cartilage development and bone healing. Some proteins involved in acute-phase or response to inflammation (Apolipoprotein A-II2, ATIII and SERPINA1) were only observed under the influence of Mg-2Ag and Pure-Mg, making them interesting for further research.

Mg-10Gd and Pure-Mg showed beneficial effects on chondrogenesis and maintenance of cartilage integrity. First, LGALS3, which has a role in the regulation of extrinsic apoptotic signalling pathway via death domain receptors, was absent. PTGS2 (having roles previously described in apoptosis, angiogenesis and bone formation) was remarkably upregulated with the three extracts, but significantly lower with Pure-Mg and Mg-2Ag than with Mg-10Gd. In the second place, the downregulation of calpain 1 (CAPN1), supported by the decreased expression of calmodulin 1 (phosphorylase kinase, delta) (CALM1) (Appendix A), which expression has been reported to diminish during chondrogenic differentiation of stem cells [62], indicate that Mg-10d and Pure-Mg extracts could enhance cell chondrogenesis. This idea is also supported by the absence of CAPN1 protein in the presence of Mg-10Gd and Pure-Mg. The serum concentration of CAPN1 raises several folds during an acute phase response (the systemic answer to a local inflammatory stimulus). Therefore its absence suggests a lack or decrease of immunological reactions against the degradation products of the material [63].

To conclude, various regulated proteins were identified in response to Mg-alloy degradation products. Regulation of specific proteins indicate a positive effect on chondrogenesis (i.e., integrins, TGFBI, FN1, VTN, CALM1, NONO) and cell viability (apoptotic-related proteins), as well as possible influence on reducing or inhibiting OA (cholesterol metabolism-related proteins and SERPINE2) and acute-phase response (APOA2, ATIII and SERPINA1). These results show that the Mg-based materials have potential to stimulate cartilage in vitro. Further investigation in vivo will be pursuit to validate these results.

Author Contributions

A.M.S. and F.F. designed the study; A.M.S, M.O., M.W., M.M., and B.L. conducted cell cultures, processing, and data analysis; H.S., R.W.R. and B.L. provided intellectual contributions; A.M.S. and M.O. cowrote the paper.

Conflicts of interest

None.

Appendix A. Supplementary data

The following is the supplementary data related to this article: