Comprehensive Biological Evaluation of Biomaterials Used in Spinal and Orthopedic Surgery

Biological acceptance is one of the most important aspects of a biomaterial and forms the basis for its clinical use. The aim of this study was a comprehensive biological evaluation (cytotoxicity test, bacterial colonization test, blood platelets adhesion test and transcriptome and proteome analysis of Saos-2 cells after contact with surface of the biomaterial) of biomaterials used in spinal and orthopedic surgery, namely, Ti6Al4V ELI (Extra Low Interstitials), its modified version obtained as a result of melting by electron beam technology (Ti6Al4V ELI-EBT), polyether ether ketone (PEEK) and polished medical steel American Iron and Steel Institute (AISI) 316L (the reference material). Biological tests were carried out using the osteoblasts-like cells (Saos-2, ATCC HTB-85) and bacteria Escherichia coli (DH5α). Results showed lack of cytotoxicity of all materials and the surfaces of both Ti6Al4V ELI and PEEK exhibit a significantly higher resistance to colonization with E. coli cells, while the more porous surface of the same titanium alloy produced by electron beam technology (EBT) is more susceptible to microbial colonization than the control surface of polished medical steel. None of the tested materials showed high toxicity in relation to E. coli cells. Susceptibility to platelet adhesion was very high for polished medical steel AISI 316L, whilst much lower for the other biomaterials and can be ranked from the lowest to the highest as follows: PEEK < Ti6Al4V ELI < Ti6Al4V ELI-EBT. The number of expressed genes in Saos-2 cells exposed to contact with the examined biomaterials reached 9463 genes in total (ranging from 8455 genes expressed in cells exposed to ELI to 9160 genes in cells exposed to PEEK). Whereas the number of differentially expressed proteins detected on two-dimensional electrophoresis gels in Saos-2 cells after contact with the examined biomaterials was 141 for PEEK, 223 for Ti6Al4V ELI and 133 for Ti6Al4V ELI-EBT. Finally, 14 proteins with altered expression were identified by mass spectrometry. In conclusion, none of the tested biomaterials showed unsatisfactory levels of cytotoxicity. The gene and protein expression analysis, that represents a completely new approach towards characterization of these biomaterials, showed that the polymer PEEK causes much more intense changes in gene and protein expression and thus influences cell metabolism.


Sample
Gene Name

Pro-MCH
Pro-MCH is proteolytically processed in the brain and in peripherial organs generating multiply protein products. Main neuropeptides are melaninconcentrating hormone (MCH), neuropeptide-glutamic acid-isoleucine (NEI), and neuropeptide-glycine-glutamic acid (NGE) that play diverse physiological functions, including energy homeostasis.
Signal transduction GPR182 G-protein coupled receptor 182 A seven-transmembrane protein that belongs to the family 1 of G-protein coupled receptors, probably acting as a receptor for adrenomedullin -a vasodilator peptide hormone expressed by all tissues, widely distributed in the cardiovascular system.
Sympathetic nerve pathway UQCRFS1 Cytochrome b-c1 complex mitochondrial subunit Rieske A component of ubiquinol-cytochrome C reductase dimer complex. As a respiratory chain, this complex generates energy essential for ATP synthesis in mitochondria.
Metabolism, respiratory electron transport SNX18 Protein sorting nexin-18, containing phox domain Members of sorting nexin family are important during progression and completion of mitosis. In case of any deficiencies in the expression of those proteins, the cytokinesis are inhibited and as a result the cell cannot finish the division. SNX18 protein was also identified as a positive regulator of autophagosome formation, that during macroautophagy fuses with lysosome. Expendable cytoplasmic constituents are then degraded and recycled within the autolysosome.
Metabolism, respiratory electron transport Autophagy GFRA2 GDNF family receptor alpha-2 GDNF (glial cell line-derived neurotrophic factor family) that constitute glycosylphosphatidylinositol (GPI)-linked cell surface receptor for GDNF and NTN. Other important function of GDNF receptor alpha-2 is activation of RET tyrosine kinase receptor.
Metabolism, respiratory electron transport LOC1027234 29 Uncharacterized gene LOC102723429 situated on short arm of 16th chromosome No further knowledge is available.

MAP2K6
Dual specificity mitogen-activated protein kinase 6 Plays an important role in signal transduction pathway. It phosphorylates a threonine and a tyrosine residue in p38 MAP kinase during different kinds of cellular stress or in response to inflammatory cytokines. Changed activity of this class of proteins may result in higher activity of NF-κB-a transcription factor regulating balance between cell survival and cell death. Dysregulation of its function may result in activation of genes involved in cancerous processes. This gene is upregulated in different kinds of cancer. Plays an oxidoreductase function but no further knowledge is available.

Signal transduction Retinoic Acid biosynthesis pathway
Signalling by Retinoic Acid (RA)

PROM2
Prominin 2 a member of prominin family Pentaspan membrane glycoprotein. It has five transmembrane segments. It is mainly expressed in epithelial cells. It binds cholesterol and plays a pivotal role in organization of membrane microdomains, such as membrane protrusions essential in diverse biological processes like cell migration and adhesion, wound healing or sensing external environment. It inhibits caveolae formation due to inhibition of CDC42 (involved in the organization of the actin cytoskeleton and endocytosis). Together with overexpression of prominin-2, larger number of membrane protrusions was observed as well as changes in PM organization.

TOMM20L ↓
Translocase of outer mitochondrial membrane 20 like Participate in assembly of translocase membrane complex responsible for passing of proteins into mitochondrial compartments. Large part of proteins needed for proper mitochondria function is encoded by nucleus of the cell thus those proteins must be transported through mitochondrial membrane to the intermembrane space. Mitochondrial membrane is impermeable for molecules larger than 5 kDa, thus proper functioning of TOM complex is crucial for mitochondria.

ELI
XLOC_l2_00 8203 ↑ RNA gene, belonging to the long noncoding RNA class.
No further knowledge is available.

SPAG9
C-Jun-N-terminal kinase-interacting protein 4 (other name: sperm associated antigen 9) Belongs to the group of scaffold proteins mediating the activation of JNK signaling pathway that controls different cellular processes like proliferation or apoptosis. It was shown that overexpression of SPAG9 gene is present in different kinds of cancer including lung, ovarian, breast and gastric cancer. The protein product probably influence tumor growth and development however up to now, its exact role in this process is still obscure.
Resulting protein participates in signal transduction but its exact role is still unknown.
Signal transduction by Rho GTPases Gene expression, Constitutive signaling in diseases SCRG1 ↓

Stimulator of chondrogenesis 1
It is associated with neurodegeneration and may participate in host response to prion infections. It was shown that this protein may influence mesenchymal stem cells differentiation during bone regeneration by interacting with bone marrow stromal cell antigen 1

USP14
Ubiquitin specific peptidase 14 It release the ubiquitin from proteins designated to be transported to proteasome. It regulates the function of proteasome and have a pivotal role in the development of human carcinoma.
Metabolism of proteins, Deubiquitinination POMZP3 POM121 and ZP3 fusion protein The protein participate in acrosin binding-a major proteolytic enzyme of mammalian spermatozoa, present in acrosome. This protein functions as a positive regulator of acrosome reaction, thus influencing the process of fertilization. It is acting as a receptor for brain injury-derived neurotrophic peptide (BINP). The role of BINP is to support of neuron survival and protection of hippocampal neurons in primary cultures from glutamate-induced cell death.

SMARCA4 Transcription activator BRG1
A protein that is a part of a large complex SNF/SWI and regulates the activation of certain genes by rearrangement of chromatin structure. This protein can play its function due to helicase and ATPase activity. What is more it can bind to BRCA1 and also influence the expression of tumorigenic protein CD44 Signal transduction, Developmental biology, nervous system development Gene expression by RUNX1 Chromatin organization

MMP14 Matrix metallopeptidase 14
A protein belonging to the MMP family involved in various physiological processes -e.g., tissue remodelling, embryonic development or in pathological conditions like arthritis and metastasis. It participates in collagen degradation, and also in skeletal connective tissue modelling. It was established that this protein activates MMP2 protein and together they are involved in formation of fibrovascular tissues. According to literature the activity of MMP2 is related to tumour invasion.
Extracellular matrix organization, collagen degradation, activation of matrix metalloproteinases

MTRNR2L2
Humanin-like 2 a peptide of 24 amino acids It was shown that this protein plays a neuroprotective role and suppress Alzheimer Disease-related neurotoxicity in vivo. It demonstrates an antiapoptotic activity of humanin in vitro-it inhibits the executive phase of apoptosis by interaction with Bax protein (an apoptosis-inducing protein) preventing its translocation from cytosol to mitochondria and thussuppressing cytochrome C release. Is present in Golgi apparatus and has a transferase activity that transfer sulfo group to glucosamine residue in heparan. Also plays role in anticoagulant heparan sulfate biosynthesis and conversion. Heparan sulfate is a linear polysaccharide that exhibit multiple biologic activities. It may bind with variety of protein ligands and form a proteoglycan (HSPG) that participates in regulating various biological processes, e.g., angiogenesis, blood coagulation, metastasis.
Metabolism, glycosaminoglycan metabolism ADAMTS1 ADAM metallopeptidase with thrombospondin type 1 motif 1 A protein belonging to the ADAMTS family, having in its structure metalloproteinase domain, disintegrin-like domain, and thrombospondintype motif. Exhibit antiangiogenic activity, expressed during process of inflammation; is also involved in development of cancer cachexia.
Metabolism of proteins, glycosylation Extracellular matrix organisation Degradation, collagen synthesis and modification, Disease (Peters-plus syndrome)

IL6ST
Interleukin 6 signal transducer A part of membrane-bound cytokine receptor complex. Acts as a signal transducer for IL6, LIF, OSM, CNTF, IL11, CTF1 and BSF3. Binding a substrate causes homodimerization of IL6SST which in turn activate Janus kinase, that phosphorylates tyrosine residues on Il6ST and finally activates STAT3 (transcription activator).
Immune system interleukin 6 and 12 signalling SNORD12C ↓ Small nucleolar RNA, C/D box 12C It contains C (UGAUGA) and D (CUGA) box sequence motifs that are highly conserved. The main function of snRNAs from box C/D family is to participate in process of RNA methylation. SNORD12C is predicted to play role during 2′ O-ribose methylation of the ribosomal 28S RNA subunit.

APH1A
Aph-1 homolog A, gamma-secretase subunit That is a non-catalytic component of gamma secretase complex (endoprotease complex) responsible for cleavage of integral membrane proteins playing important role in Notch or Wnt signalling cascades. It participates also in APP (amyloid-beta precursor protein) processing. It was shown that APH-1A gene polymorphism contribute to increased incidence of Alzheimer Disease. It was shown that downregulation of FOXD3-AS1 in malignant glioma cells has an inhibitory effect on cell proliferation, migration and invasion. Upregulation of this RNA gene results in poorer prognosis and lower survival probability for glioma patients. It was stated that FOXD3-AS1 has a tumorigenic property that may cause development of glioma. Another study demonstrated that this non-coding RNA is highly expressed in breast cancer-and may serve as a prognostic biomarker for breast cancer patients.
Developmental biology, transcriptional regulation of stem cells A_22_P0002 0320 Not annotated No further knowledge is available.

PPA2
Inorganic pyrophosphatase 2 A protein present in mitochondria that catalyze the pyrophosphate hydrolysis to the inorganic phosphate. Impaired function of this hydrolase can result in deregulation of mitochondrial membrane potential, affecting proper function of whole mitochondrium. It is also essential for proper cell metabolism. Mutation in PPA2 gene is correlated with occurrence of mitochondrial diseases and cardiomyopathy.
Metabolism, phyrophosphate hydrolysis NLE1 Notchless homolog 1 A protein that takes part in regulation of CDKN1A expression and other proteins involved in Wnt signal transduction pathways. In Wnt signaling pathways signals are passed to the cell through cell surface receptors and are important for normal cell functioning, but also impaired activity of this pathway can contribute to cancer spreading.

ELI-EBT TMEM65
↑ Transmembrane protein 65 A mitochondrial inner-membrane protein. It was demonstrated that this protein plays a significant role in mitochondrial respiratory chain. Mutation in TMEM65 results in mitochondrial dysfunction and clinically is demonstrated by encephalomyopathy. This protein is also suspected for participating in maintaining mitochondrial DNA stability.
Metabolism of lipids estrogen, cholesterol synthesis Immune system, interleukin 3, 5 and GM-CSF signaling Organelle biogenesis and maintenance Cell-cell communication Nectin/Necl trans heterodimerization Metabolism, complex I biogenesis and electron transport Transport of small molecules CPA4 ↓ Carboxypeptidase A4 A metalloprotease belonging to a carboxypeptidase A/B subfamily, that catalyses proteolysis of C-terminal amino acids. It plays a probable role in histone hyperacetylation and also is considered as a cancer related gene. Increased expression of CPA4 protein was found in cancer tissue derived from patients suffered from colorectal cancer. It was shown that the presence of elevated level of this protein was corelated with poorer prognosis. In vivo studies confirmed tumorigenic function of this protein due to activation of STAT3 and ERK pathways (the first one act as a signal transducer and activator of transcription, the later -is a mitogen activated protein kinase that regulates different cellular processes like autophagy, protein trafficking and integrity of genome). What is more, group of Sun et al. has observed that higher expression of CPA4 was a good prognostic parameter for prediction of hepatic metastasis, its progression and stage.

ANKRD1
Ankyrin repeat domain 1 A protein involved in activation of endothelial cells. It is localized in cell nucleus and may play role of transcription factor that regulates cardiac gene expression. It is also linked to the epithelial-mesenchymal transition (EMT) during cancer development. It was shown that ANKRD1 is overexpressed in EGFR-TKIs-resistant nonsmall cell lung cancer (NSCLC; EGFR-TKIs -epidermal growth factor receptor tyrosine kinase inhibitors). ANKRD1 gene silencing caused decreased resistance of cancer cells towards therapeutic drugs-afatinib and osimertinib. Inhibition of ANKRD1 expression at mRNA or protein level can be a promising therapeutic target for EGFR-mutant NSCLC patients. Implicated in mitochondrial protein import and macromolecular assembly.