Identification of New Predictive Osseointegration Bio-Markers of the Prosthetic Implant in Patients With OsteoArthritis
Conditions: Osteoarthritis
Sponsor: Istituto Ortopedico Rizzoli

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Identification of new Predictive Osseointegration Bio-Markers of the 
prosthetic implant in patients with OsteoArthritis" 
 
Study Code: Bio.Pre.I.OA 
Sponsor: 
IRCCS - Istituto Ortopedico Rizzoli 
Via di Barbiano 1/10 
40136 Bologna 
Italy 
Version and date Vers 1.1 
24/04/2018 
Coordinator Center: 
Istituto Ortopedico Rizzoli 
Piattaforma Tecnologica per l’Ingegneria Tissutale, 
Teranostica ed Oncologia, Dipartimento Rizzoli-Sicilia 
SS 113 km 246 
90011 Bagheria (PA) 
Principal Investigator: 
Dott.ssa Viviana Costa 
Tel: 091-6554630 
E-mail : viviana.costa@ior.it 
Study Laboratory: 
Piattaforma Tecnologica per l’Ingegneria Tissutale, 
Teranostica ed Oncologia, Dipartimento Rizzoli-Sicilia 
Via Divisi, 83 Palermo 
90133 Palermo 
Study methodology: In vitro biological study with human derived matererial  
Type: No profit 
Funding: None 
 

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TABLE of CONTENTS 
Background and study rational   
 
 
 
 
 
            pg. 3 
Objectives of the study 
 
 
 
 
 
 
            pg.  4 
Study design   
 
 
 
 
 
 
                         pg.  4 
Population  
 
 
 
 
 
 
 
 
            pg.  4 
Material and Methods 
 
 
 
 
 
 
            pg.  5 
Statistics 
 
 
   
 
 
 
 
 
            pg.  6 
Enrollment Procedures 
  
 
 
 
 
 
            pg.  6 
Data Collection           
  
 
 
 
 
 
            pg.  6 
Ethics                     
  
 
 
 
 
                                      pg.  7 
Informed Consent  
 
  
 
 
 
 
 
            pg.  7 
General principles for human biological material (HBM) collection                         pg. 7 
Confidentiality                                                                                       
 
 
pg.  8 
Publication policy and data ownership                                                                          pg.  8                                               
References                                                                                       
 
            pg.  9 

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Background and study rational   
Age-related degenerative disorders, such as osteoarthritis, are among the diseases with the greatest socio-
economic impact; moreover, the increase in life expectancy of the population has led to an increase in the 
number of people affected by this disease in the last decades [Pelletier et al., 2001]. Osteoarthritis (OA) is a 
chronic joint degenerative disease characterized by degeneration of hyaline articular cartilage, synovial 
inflammation and changes in the subchondral bone. There are two forms of OA, primary and secondary; 
the latter occurs at a later age, tending after 60 years, and is due to the natural process of aging of the joint 
and / or to events of a traumatic nature. Several other etiopathogenetic factors are involved in the 
development of this degenerative process including genetic factors, obesity and chronic inflammatory 
status, and the complex pathogenesis is not yet fully determined. The joints most affected are those of the 
upper and especially lower limbs and the spine and the symptoms are algia, joint stiffness, muscle tension 
and instability, which induce the patient to immobility in an attempt to avoid pain; this symptomatology 
often aggravates the quality of life of elderly patients to produce a depressive state that further 
complicates the general clinical picture [Pelletier et al 2001; Hunziker, 2001]. The current pharmacological 
and physiatric treatments are not resolutive of OA and in fact, the prosthetic intervention turns out to be 
the only solution for the patient. In recent decades, the technology has developed more and more 
performing joint prostheses, inspired by different mechanical and biological principles, in order to better 
reproduce joint physiology and improve the efficiency of the implant. Currently the materials used have a 
long life span, and 90 to 95% of the operated patients have a prosthetic implant survival 10 to 15 years 
after surgery. However, implants can also fail for several reasons: dislocation, aseptic loosening, peri-
prosthetic infections aggravated by sometimes predisposing factors of the patient, such as diabetes, which 
determine the need to perform a revision surgery [Pelletier et al., 2001 ]. In spite of this, chondrocyte has a 
key role in the degradation of hyaline cartilage by: i) an imbalance between its own anabolic and catabolic 
activity; and ii) to the reduction of the chemical-physical properties, other cellular components are more 
involved in the subsequent possible failure of the integration of the prosthetic implant. Modifications of the 
joint microenvironment, the deficiency of the regenerative response of the osteoblasts, the inappropriate 
activation of the chemochemical network and the activation of the synoviocytes in the inflammatory sense 
(activation of the pathways of NF-KB, HIF1, BMP, TGF-β, and IGF) they are all conditions and factors that 
can alter the osseointegrative process of a joint prosthesis [Pelletier et al 2001; Hunziker, 2001; Asahara H. 
et al., 2016]. The aim of the present study is to identify and evaluate the possible role of miRNAs and long 
non-coding RNA (lncRNA), modulated in OA, as new biomarkers useful to predict the efficiency of the 
osseointegration process of the prosthetic implant [Xu et al. 2016; Zhang et al. 2017; Bernard NJ. et al., 
2014]. 
 
 

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Objectives of the study  
 
Primary Objective 
Identification and characterization of miRNAs and LncRNA, from synoviocytes, osteoblasts and 
chondrocytes 
isolated 
from 
tissue 
samples 
obtained 
from 
endo 
or 
surgery 
artro-prosthesis, and related signaling involved in synoviocytes and osteoblasts cross-talkduring the 
osteointegration 
process 
through 
suitable 
in 
vitro 
co-culture 
models 
[Craig 
I.Jet al., 2016; Lin Y.et al., 2010]. 
 
Secondary Objectives: 
Identification of other biomarkers useful for predicting the efficiency of osseointegration of the 
implant, through bioinformatics and molecular analysis of miRNAs, lncRNA and proteins secreted or 
produced by synoviocytes and osteoblasts; 
 
Validation of data obtained on miRNAs and lncRNAs through "gain and loss of" experiments 
function "in primary cultures and co-cultures. 
 
Study design  
The study involves the isolation and cellular, molecular and proteomic characterization of synoviocytes, 
osteoblasts and chondrocytes from “waste” tissue samples obtained from patients undergoing surgery of 
endo or arthroplasty for osteoarthritis (Experimental Group) or interventions for other causes such as for 
example periarticular fracture requiring the implantation of a prosthesis (Control Group) at the Rizzoli-
Sicilia Department (Bagheria-Palermo).  
Deeper analysis of miRNAs and lncRNAs expressed by the synoviocytes, osteoblasts and chondrocytes will 
be performed in order to understand their possible involvement in the signaling of HIF1-, -catenin, NF-Kb 
and other pathways of osteogenic differentiation, through suitable experimental in vitro models of co-
cultures. In particular, we will focus on the cross-talk between synoviocytes and osteoblasts, cell 
populations involved in the post-surgical articular microenvironment and on which the osteointegration 
process of the joint prosthesis depends. 
 
Population  
Inclusion Criteria: 
1) Male and Female patients > 40 years old hospitalized for surgery of endo- or arthroplasty for OA 
(Group OA) or for surgery for joint traumatic diseases (eg femoral neck fractures) requiring the 
implantation of a prosthesis (Control Group); 
2) Interval of at least three months from any previous infiltrative treatment of any kind; 

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3) Patients able to provide written informed consent to the study. 
Exclusion Criteria 
1) Severe cognitive defects or psychiatric disorders; 
2) Tumor pathologies or concurrent antineoplastic therapies; 
3) Autoimmune diseases (rheumatoid arthritis); 
4) Surgery that involves the joint 
 
Material and Methods  
The isolated cells (synoviocytes, osteoblasts and chondrocytes) will be kept in culture under specific 
conditions in order to allow the isolation of proteins, mRNAs, miRNAs and lncRNAs. Cellular 
characterizations will be performed using FACS (Partec CyFlow Space), while the molecular 
characterizations will be obtained with qRT-PCR (Light-Cycler 2.0-Roche). Protein / proteomic analysis will 
be performed using ELISA (commercial kit), Western blot (ChemiDoc XRS + System, BIORAD) and TRIPLE 
TOF5600 (AB Sciex). The miRNAs and lncRNAs, identified by molecular biology techniques (qRT-PCR, LINE 
GENE 9640), the mRNAs and the identified proteins will be analyzed through the use of specific software, 
such as: TargetScanPrediction, Diana Software, MiRBase and Partec FloMax®. The miRNAs and lncRNAs that 
will be particularly regulated / modulated following the pathology and the reduced activation of the 
pathways involved in osteoblastic differentiation, will be investigated, evaluating their involvement in the 
regulation of HIF1-, -catenin, NF-Kb signaling and other possible pathways of osteogenic differentiation. 
Through in vitro co-culture systems (osteoblasts and synoviocytes isolated from tissues), we will evaluate 
the role of previously identified miRNAs and lncRNAs, in cross-talk between osteoblasts and synoviocytes 
isolated from patients with OA. Their role will be validated through "gain and loss of function" experiments 
in primary cultures and co-cultures. 
 
Statistics  
Multivariate statistical analysis of the results will be performed using R software [R Development Core 
Team (2008) software. A: A language and environment for statistical computing. R Foundation for Statistical 
Computing, Vienna, Austria. ISBN 3-900051-07-0, URL http://www.R-project.org.] After evaluating the 
normal distribution homogeneity of the variance of data the appropriate parametric statistical tests will be 
applied (ANOVA or GLM and Student t test) or in case of non-normality and heteroskedasticity similar non-
parametric tests (eg Kruskal-Wallis and Mann-Whitney U test). The proteomics data will be analyzed by 
principal components analysis  using  MarkerViewTM 1.2.1 (SCIEX) software. The molecular interaction 
network between the modulated proteins that are found to be significant will be analyzed with the STRING 
software v10 (Search Tool for the Retrieval of Interacting Genes / Proteins; http://string-db.org/). The 
sample size was calculated using the G*Power software v.3.1.9.2 (University of Kiel, Germany), considering 

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a significance level α = 0.05 and a power 1-β = 0.80. For the calculation of the effect size we considered the 
result of variation of ALPL gene expression in human mesenchymal stem cells cultured in hypoxic 
environment (similar to the intra-articular environment) between 3 (FOI: 1.53 ± 0.34) and 7 (FOI: 2.53 ± 
0.29) days and presenting an increased expression of miR-675-5p [Costa V. et al, 2017]. The effect size 
value d=2.28 was transformed into f=1.14, because the sample size was calculated considering a two-way 
ANOVA model (patient: healthy / osteoarthritis, experimental culture time: 2 short times). The maximum 
number of patients to be enrolled is n=10 of which n=5 osteoarthritic and n=5 healthy. 
 
Enrollment Procedures  
Patients considered eligible will be enrolled in the study, after providing a written informed 
consent.  
 
Data Collection 
Clinical data will be retrieved by patient’s source document 
A protocol-specific CRF reporting the results of the analyses will be provided 
A CRF is required and should be completed for each included subject. 
 
Ethics 
The clinical trial protocol and its documents will be sent before initiating the study to the 
competent Authorities and Ethics Committees of each participating country for its approval. 
The responsible investigator will ensure that this study is conducted in agreement with either the 
most updated Declaration of Helsinki and all the international and local laws that apply to clinical 
trials and to patient protection.  
The protocol has been written, and the study will be conducted according to the principles of the 
ICH Harmonized Tripartite Guideline for Good Clinical Practice  
(ref: http://www.emea.eu.int/pdfs/human/ich/013595en.pdf).  
 
Informed Consent 
All patients will be informed, by the investigator, of the aims of the study, the possible risks and 
benefits that will derive from the study participation.  
The Investigator must clearly inform that the patient is free to refuse participation in the study and 
that can withdraw consent at any time and for any reason.  

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They will be informed as to the strict confidentiality of their patient data, but that their medical 
records may be reviewed for trial purposes by authorized individuals other than their treating 
physician.  
The informed consent procedure must conform to the ICH guidelines on Good Clinical Practice. 
This implies that "the written informed consent form should be signed and personally dated by the 
patient or by the patient's legally acceptable representative".   
The Investigator must also sign the Informed Consent form, and will keep the original at the site 
and a copy of the original must be handed to the patient.  
The competent ethics committee for each Institution participating to the study must validate local 
informed consent documents before the study can be opened. It will be emphasized that the 
participation is voluntary and that the patient is allowed to refuse further participation in the 
study whenever he/she wants. This will not prejudice the patient's subsequent care.  
 
General Principles for Human Biological Material (HBM) collection 
Human biological material (HBM) collection involves the collection and storage of biological 
material, residual biological material or derivatives in compliance with ethical and technical 
requirements.  
Biological material ( tissue samples) will be centralized and stored at l’Istituto Ortopedico Rizzoli - 
Dipartimento Rizzoli-Sicilia, Bagheria (Palermo). 
From here, the biological material will be used and stored according with the sample characteristic 
and applicable regulation. 
The molecular and protein materials obtained from the isolated cells will be stored inside sterile containers 
(falcon, tubes, eppendorf) in the freezer at the temperature of -80 ° C at the Istituto Ortopedico Rizzoli -
Dipartimento Rizzoli Sicilia. 
The “Piattaforma Tecnologica per l'Ingegneria Tissutale, Teranostica ed Oncologia” laboratory will 
perform the research as stated in the protocol “Material and Methods” section  
The following principles apply to storage of HBM: 
 The Istituto Ortopedico Rizzoli will have a designated person responsible for collection and 
will act as a communication point  
 The collected HBM should be documented, i.e. the amount remaining and its location. act 
as a communication point  
 The storage and use of biological material will take place in accordance with the standards 
of good laboratory practice (GLP) and applicable legislation. 
 

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Confidentiality 
In order to ensure confidentiality of clinical trial data as disposed the national and European 
applicable regulation, data will be only accessible for the trial Sponsor and its designees, for 
monitoring/auditing procedures, the Investigator and collaborators, the Ethics Committee of each 
corresponding site and the Health Authority. 
Investigator and the Institution will allow access to data and source documentation for 
monitoring, auditing, Ethic Committee revision and inspections of Health Authority, but 
maintaining at all times subject personal data confidentiality as specified in the applicable 
regulation. 
 The Investigator must guarantee that patient anonymity is kept at all times and their identity 
must be protected from unauthorized persons and institutions.  
All patients included in the study will be identified with a numeric code, so that no identifiable 
personal data will be collected. 
The Investigator must have and conserve a patients’ inclusion registry where it figures the 
personal data of the patient: name, surname, address and corresponding identification code into 
the study, this register will be kept on the Investigator File. 
 
Publication policy and data ownership  
The principal investigator of the study is responsible for the final report, of the publication publish all the 
data collected as described in the protocol and will ensure that the data are reported responsibly and 
consistently. 
In particular, the publication of data deriving from the present study will take place independently of the 
results obtained. 
The transmission or dissemination of data, through scientific publications and / or presentation at 
conferences, congresses and seminars, will take place exclusively following the purely statistical processing 
of the same, or in an absolutely anonymous form 
All the study data are owned by the sponsor. 
 
References 
1. Pelletier, J.P.; Martel-Pelletier, J.; Abramson, S.B. Osteoarthritis, an inflammatory disease: 
Potential implication for the selection of new therapeutic targets. Arthritis Rheumatol. 2001, 
44, 1237–1247 

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2. Hunziker EB. Articular cartilage repair: basic science and clinical progress. A review of the 
current status and prospects. Osteoarthritis Cartilage 2002; 10(6): 432-63. 
3. Zhang M., Kate Lygrisse and Jinxi Wang. Role of MicroRNA in Osteoarthritis. J Arthritis. 2017 
Apr; 6(2): 239. 
4. Xu B.,Li Y., Ma J., and Fu-xing Pei. Roles of microRNA and signaling pathway in osteoarthritis 
pathogenesis. J Zhejiang Univ Sci B. 2016 Mar; 17(3): 200–208. 
5. Craig I.J., Argyle D.,Clements N. In vitro models for the study of osteoarthritis. Vet J. 2016 
Mar;209:40-9. doi.org/10.1016/j.tvjl.2015.07.011. 
6. Lin Y.Y., N. Tanaka, S. Ohkuma, Y. Iwabuchi, Y. Tanne, T. Kamiya, R. Kunimatsu, Huang Y.-C., M. 
Yoshioka, T. Mitsuyoshi, et al. Applying an excessive mechanical stress alters the effect of 
subchondral osteoblasts on chondrocytes in a co-culture system. European Journal of Oral 
Sciences, 118 (2010), pp. 151-158. 
7. Akhtar N, Rasheed Z, Ramamurthy S, Anbazhagan AN, Voss FR, Haqqi TM. MicroRNA-27b 
regulates the expression of matrix metalloproteinase 13 in human osteoarthritis 
chondrocytes. Arthritis Rheum. 2010 May;62(5):1361-71. doi: 10.1002/art.27329. 
8. Bernard NJ. Osteoarthritis: circulating miRNAs-early osteoarthritis biomarkers? Nat Rev 
Rheumatol. 2014 Apr;10(4):197. doi: 10.1038/nrrheum.2014.25. Epub 2014 Feb 25. 
9. Asahara H. Current Status and Strategy of microRNA Research for Cartilage Development and 
OsteoarthritisPathogenesis. 
J 
Bone 
Metab. 
2016 
Aug;23(3):121-7. 
doi: 
10.11005/jbm.2016.23.3.121. Epub 2016 Aug 31. Review. 
10. Alcaraz MJ, Megías J, García-Arnandis I, Clérigues V, Guillén MI.New molecular targets for the 
treatment 
of 
osteoarthritis. 
Biochem 
Pharmacol. 
2010 
Jul 
1;80(1):13-21. 
doi: 
10.1016/j.bcp.2010.02.017. Epub 2010 Mar 3. Review 
11. Costa V, Raimondi L, Conigliaro A, Salamanna F, Carina V, De Luca A, Bellavia D, Alessandro R, 
Fini M, Giavaresi G. Hypoxia-inducible factor 1Α may regulate the commitment of 
mesenchymal stromal cells toward angio-osteogenesis by mirna-675-5P. Cytotherapy. 
2017;19:1412-1425.].