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How early inflammatory events affect bone nano-properties at the onset of rheumatoid arthritis
Sumary Rheumatoid arthritis (RA) is a chronic, inflammatory, autoimmune disease mainly characterized by joint and bone destruction. Based on previous results in an arthritis mouse model, suggesting a direct effect of inflammation on bone mechanical properties and collagen turnover and organization, it is tentative to hypothesize that the initial steps towards bone fragility are determined by early changes in collagen type I organization capable of interfering with the intrinsic bone tissue properties. These are bone nano level properties, independent of the overall bone architecture and directly dependent on the way bone cells, collagen and calcium crystals interact. The main goal of this project is to identify, in the very early phase of arthritis, using an animal model, the leading changes in nano bone structure. In addition, the presence of these disturbances will be tested, in latter stage arthritis, in human RA bone samples.
Objectives
In our Rheumatology Research Unit we have been developing translational research in the field of rheumatoid arthritis and bone.
The main goal of my project is to verify, in the very early phase of arthritis, how inflammation affects bone cellular activity and collagen type I organization and its interference with the intrinsic bone tissue properties.
Specific aim 1: I will specifically verify the influence of arthritis on: a) the micro structure of bone; b) the degree of mineralization and mineralization distribution; c) the fine arrangement of collagen molecules and crystals; d) the mechanical properties at a tissue level; e) the cellular activity.
Specific aim 2:
Using a TNF antagonist drug I will verify if early treatment intervention will abrogate the effects verified in aim 1.
Specific aim 3: Using the information gathered with the animal model, I shall identify the bone disturbances that actually occur in RA patients, assessing samples from joint surgery through the same technical procedures listed above.
Identifying early bone cell and collagen disturbances as the first step for joint and bone failure in RA could change the management strategy of this disease, emphasizing the need for early control of inflammatory activity but also stressing the relevance of controlling bone intrinsic properties, upon disease onset, through innovative bone specific treatment approaches that could be based in concepts developed by this project.
Animal model studies
In this project I will use the rat adjuvant-induced arthritis model to study the effects of inflammation on bone in the very early phase of arthritis.
The findings from the arthritic group will be compared to a control group without induced arthritis.
Blood samples will be collected to measure bone turnover markers (P1NP and CTX) and cytokines levels (TNF, RANKL, OPG, IL1, IL17 and IL6).
Bone samples will be submitted to several technical procedures:
a) Micro-computed tomography (micro-CT) will be performed. Based on hundreds of angular views acquired while the sample rotates, allowing the acquisition of a 3D image, American Society for Bone and Mineral Research parameters of bone micro structure will be obtained.
b) The overall bone structure, degree of mineralization and osteoid, will be also assessed by conventional histology and histomorphometry.
c) Mineralization distribution pattern will be complemented by quantitative backscattered electron imaging.
e) The structure and fine arrangement of collagen molecules and crystal distribution will be assessed by atomic force microscopy (AFM). Nanoidentation will test the intrinsic mechanical characteristics of bone tissue.
f) Bone cell function and RNA expression will be analyzed.
An early treatment intervention with a TNF antagonist drug will be performed to assess the early effects of this treatment on the bone effects of arthritis previously characterized by us.
Human samples assessment
The same procedures will be applied to human bone samples from rheumatoid arthritis patients submitted to total hip replacement surgery.
During the last 4 years, our unit has established a collaboration with Hospital Santa Maria Orthopedic Department and all femoral heads removed from patients submitted to joint replacement surgery are being sent to us for research purposes. For this project all consecutive patients submitted to hip arthroplasty in the Orthopaedic Department of HSM will be evaluated for potential inclusion.
After giving their written informed consent, patients will be interviewed in order to collect clinical data according to a previously published questionnaire and a dual energy x-ray absorptiometry (DXA) scan will be performed. Blood and urine samples will be collected for determination of laboratorial bone metabolic parameters, inflammatory and bone turnover markers.
Based on the statistics of the Orthopaedic Department and in our previous collaborative work we expect to recruit 300 femoral heads during 24 months and that around 20 will be from RA patients. Clinical assessment of RA patients, Disease Activity Score and Health Assessment Questionnaire will be performed. Rheumatoid factor (RF) and anti-cyclic citrullinated peptides (CCP) antibodies will be determined. From the rest of the sample we will select a control group of patients submitted to hip replacement surgery due to osteoarthritis (a non inflammatory condition, not associated with osteoporosis), matched for sex and age with the RA group, with normal bone mineral density (BMD) and absence of risk factors for fracture. The existence of any metabolic bone disease or inflammatory disease will be an exclusion criterion. We expect to recruit around 20 of these control individuals.
Given the obvious limitations for obtaining a true healthy control and taking into consideration that the evaluations that we will perform are not possible to be done in cadaveric samples, this is the best possible approach of comparing RA bone with a bone that is not affected either by inflammation or by a metabolic bone disease. A second comparison will be done with a group of patients submitted to arthroplasty due to hip fragility fracture, matched for sex, age, risk factors for fracture and BMD with the RA group. We expect to recruit around 20 of these osteoporotic individuals. This group will allow the comparison of RA samples with a matched fracture risk population that only differs in the absence of an inflammatory disease.
Bone samples of these 3 groups will be submitted to the technical procedures described above.
In summary, I expect that this work could constitute a major contribution to a frequent and debilitating disease by identifying the initial mechanisms of bone collapse in RA and providing new potential targets for future treatment, fulfilling a clinical unmet need.
Bruno Vidal
Estudante de doutoramento
Objectives
In our Rheumatology Research Unit we have been developing translational research in the field of rheumatoid arthritis and bone.
The main goal of my project is to verify, in the very early phase of arthritis, how inflammation affects bone cellular activity and collagen type I organization and its interference with the intrinsic bone tissue properties.
Specific aim 1: I will specifically verify the influence of arthritis on: a) the micro structure of bone; b) the degree of mineralization and mineralization distribution; c) the fine arrangement of collagen molecules and crystals; d) the mechanical properties at a tissue level; e) the cellular activity.
Specific aim 2:
Using a TNF antagonist drug I will verify if early treatment intervention will abrogate the effects verified in aim 1.
Specific aim 3: Using the information gathered with the animal model, I shall identify the bone disturbances that actually occur in RA patients, assessing samples from joint surgery through the same technical procedures listed above.
Identifying early bone cell and collagen disturbances as the first step for joint and bone failure in RA could change the management strategy of this disease, emphasizing the need for early control of inflammatory activity but also stressing the relevance of controlling bone intrinsic properties, upon disease onset, through innovative bone specific treatment approaches that could be based in concepts developed by this project.
Animal model studies
In this project I will use the rat adjuvant-induced arthritis model to study the effects of inflammation on bone in the very early phase of arthritis.
The findings from the arthritic group will be compared to a control group without induced arthritis.
Blood samples will be collected to measure bone turnover markers (P1NP and CTX) and cytokines levels (TNF, RANKL, OPG, IL1, IL17 and IL6).
Bone samples will be submitted to several technical procedures:
a) Micro-computed tomography (micro-CT) will be performed. Based on hundreds of angular views acquired while the sample rotates, allowing the acquisition of a 3D image, American Society for Bone and Mineral Research parameters of bone micro structure will be obtained.
b) The overall bone structure, degree of mineralization and osteoid, will be also assessed by conventional histology and histomorphometry.
c) Mineralization distribution pattern will be complemented by quantitative backscattered electron imaging.
e) The structure and fine arrangement of collagen molecules and crystal distribution will be assessed by atomic force microscopy (AFM). Nanoidentation will test the intrinsic mechanical characteristics of bone tissue.
f) Bone cell function and RNA expression will be analyzed.
An early treatment intervention with a TNF antagonist drug will be performed to assess the early effects of this treatment on the bone effects of arthritis previously characterized by us.
Human samples assessment
The same procedures will be applied to human bone samples from rheumatoid arthritis patients submitted to total hip replacement surgery.
During the last 4 years, our unit has established a collaboration with Hospital Santa Maria Orthopedic Department and all femoral heads removed from patients submitted to joint replacement surgery are being sent to us for research purposes. For this project all consecutive patients submitted to hip arthroplasty in the Orthopaedic Department of HSM will be evaluated for potential inclusion.
After giving their written informed consent, patients will be interviewed in order to collect clinical data according to a previously published questionnaire and a dual energy x-ray absorptiometry (DXA) scan will be performed. Blood and urine samples will be collected for determination of laboratorial bone metabolic parameters, inflammatory and bone turnover markers.
Based on the statistics of the Orthopaedic Department and in our previous collaborative work we expect to recruit 300 femoral heads during 24 months and that around 20 will be from RA patients. Clinical assessment of RA patients, Disease Activity Score and Health Assessment Questionnaire will be performed. Rheumatoid factor (RF) and anti-cyclic citrullinated peptides (CCP) antibodies will be determined. From the rest of the sample we will select a control group of patients submitted to hip replacement surgery due to osteoarthritis (a non inflammatory condition, not associated with osteoporosis), matched for sex and age with the RA group, with normal bone mineral density (BMD) and absence of risk factors for fracture. The existence of any metabolic bone disease or inflammatory disease will be an exclusion criterion. We expect to recruit around 20 of these control individuals.
Given the obvious limitations for obtaining a true healthy control and taking into consideration that the evaluations that we will perform are not possible to be done in cadaveric samples, this is the best possible approach of comparing RA bone with a bone that is not affected either by inflammation or by a metabolic bone disease. A second comparison will be done with a group of patients submitted to arthroplasty due to hip fragility fracture, matched for sex, age, risk factors for fracture and BMD with the RA group. We expect to recruit around 20 of these osteoporotic individuals. This group will allow the comparison of RA samples with a matched fracture risk population that only differs in the absence of an inflammatory disease.
Bone samples of these 3 groups will be submitted to the technical procedures described above.
In summary, I expect that this work could constitute a major contribution to a frequent and debilitating disease by identifying the initial mechanisms of bone collapse in RA and providing new potential targets for future treatment, fulfilling a clinical unmet need.
Bruno Vidal
Estudante de doutoramento
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