the Type ONe dIabetic Bone Collaboration Study

Diabetes Mellitus, Type 1 Clinical Trial

— TONICS  

Official title:

the Type ONe dIabetic Bone Collaboration Study

Clinical Trial Details — Status: Active, not recruiting

Administrative data

• NCT number: NCT05673837
• Other study ID #: S-20200192
• Status: Active, not recruiting
• Start date: December 10, 2021
• Est. completion date: April 1, 2024

Study information

• Verified date: October 2023
• Source: Odense University Hospital
• Contact: n/a
• Is FDA regulated: No
• Study type: Observational

Clinical Trial Summary

In this cross-sectional clinical study, we will examine the bones of 111 Type 1 Diabetes (T1D) patients and 37 age-matched healthy controls with the aim of describing a T1D Bone Phenotype. The main Objectives of the study is a) to determine if the material properties of the bones are affected in diabetic bone disease and b) to determine if the mitochondrial function in osteoclasts and osteoblasts is impaired in T1D. Secondary end points are c) to establishment of the T1D bone phenotype and d) to investigate if mitochondrial dysfunction in T1D bone cells correlates to changes in gene expression, gene activity, bone remodelling, bone density, microarchitecture, geometry and material properties. Furthermore, in terms of contributing to knowledge on etiology and pathology of type one diabetic bone disease, we will study the predictory value of muscle mass in T1D patients and controls, as well as other characteristics such as heart rate variability (HRV) and AGE content. Furthermore, we will study the epidemiology of osteoporosis and fractures in Danish T1D patients.

To assess the material properties of the bones, we will measure the bone mass density (BMD), use High Resolution peripheral Quantitative Computed Tomography (HRpQCT) for assessment of the microarchitecture and finite element analysis of bone strength, and by microindentation, we will obtain direct measures of the strength of the cortical bone of the tibia. Further we will measure bone turnover markers and circulating microRNA and in a subgroup of participants (24 T1D, 12 controls) bone samples will be retrieved for examination of bone histomorphometry (structural and static parameters) and cell samples from blood and bone marrow will be used for in vitro experiments focused on cell differentiation mitochondrial function, as hyperglycemia may affect mitochondrial function. Finally measures of some possible predictors of bone fragility in subjects with T1D are examined (sarcopenia, skin advanced glycation end products (AGE) content, autonomic neuropathy)

Description:

Methods The project consists of a cross-sectional case-control study bone histology, density and strength in adult T1D patients and in vitro studies of bone cell metabolism, including mitochondrial function in T1D.

Participants 111 male and female patients aged 18-80 years with early onset T1D (diagnosed before age of 18) and 37 healthy age-, sex- and BMI-matched control subjects. Of those 24 cases and 12 controls are needed for studies on bone biopsies and bone marrow aspirates.

Investigations Clinical data from digital medical records, survey and interview: c-peptide levels, medical history including information on diabetes-related complications, fracture history and concomitant medication.

Blood samples: hemoglobin A1c, ionized calcium (Ca2+), parathyroid hormone (PTH), 25-hydroxycholecalciferol, Hemoglobulin, Leucocytes, platelets, creatinin, estimated glomerular filtration rate (eGFR), Alkaline phosphatase, Alanine transaminase, albumin, international normalized prothrombin ratio (INR). And possibly c-peptide and T1D-related antibodies on T1D subjects, may these results not be found in the medical records. Fasting serum levels of biochemical markers of bone resorption, bone formation and inhibition of bone formation: Cross-linked C-telopeptide of type I collagen (CTX) Procollagen-1 N-terminal peptide (P1NP), Sclerostin, fasting levels of circulating miRNAs reported to be associated with bone turnover will be measured using quantitative PCR (qPCR) by use of a LightCycler 480 instrument (will be sent to measurement in Vienna (Dr M Hackl, TamiRNA, Vienna, Austria)).

For the participants that have accepted to have bone biopsy and marrow aspirate performed, we will measure Hemoglobulin, platelets, INR, activated partial thromboplastin time (APTT) 5 days prior to the biopsy. From these subjects, we will also obtain fullblood for: In vivo investigations on monocytes (osteoclastogenesis) and Resorption assays.

Dual-energy X-ray Absorptiometry (DXA) scan will provide BMD at the hip and spine as well as Trabecular Bone Score (TBS) and whole-body scan for a measure of muscle mass HR-pQCT at the distal radius and tibia, we will obtain: Bone geometry, Volumetric BMD, Bone microarchitecture, Bone strength.

by microindentation using OsteoProbe®, we will measure the material strength index (BMSi), a measure of the bone hardness and stiffness.

Bone biopsy: following bone labelling with oral tetracycline, an 8 mm trans iliac bone biopsy will be performed. Bone specimens will be plastic embedded and used for

• Bone histomorphometry (structural, static and dynamic parameters)

• Micro Finite Element Analysis (strength analysis)

• Ultra-high resolution measurements on osteocytes (number, density and connections)

• Immunohistochemistry and imaging for coupling factors and gene expression. Cryosectioned bone tissue will be used to perform spatial transcriptomics to assess the gene activity in bone-specific loci.

A Bone marrow aspirate 10-15 ml will be secured and used to study bone cells:

• Flowcytometric methods on in vitro studies on mesenchymal stem cells (MSCs) from bone marrow aspirates: expression, differentiation and proliferation to osteoblasts(OBs) and adipocytes.

• Monocytes from peripheral blood will be used to study osteoclastogenesis and bone resorption activity

• In vitro studies and proteomics of bone cell communication in co-cultures of OBs and osteoclast (OCs).

• Single cell RNA sequencing will be performed to demonstrate gene signatures of MSCs, OBs and OCs in T1D patients and controls.

• Determination of the metabolic profile (using the Seahorse Xfe96 Analyzer) and measurement of glucose uptake of MSCs, OBs and OCs in T1D patients and controls

• Imaging of mitochondrial morphology using MitoTracker.

• In situ hybridization and immunohistochemistry Outcome

Differences between early onset T1D patients and healthy age-matched controls in following parameters and the relationship between these parameters in T1D patients:

• BONE PHENOTYPE: Bone density, geometry, architecture, histomorphometry, ultra-high resolution measurements on osteocytes and finite element analysis (FEA) - calculated strength obtained from DXA scans, HRpQCT scans and from bone biopsies in a subgroup of the subjects and Bone hardness (Bone Score), measured by microindentation.

• BONE METABOLISM: Levels of skinAGEs and levels of biochemical bone turnover markers and circulating miRNA's known to be related to bone turnover, dynamic measures from bone histomorphometry. Immunohistochemistry and -imaging on bone biopsies.

In vitro analyses and other investigations on cells will produce outcomes on bone cell differentiation, proliferation and intercellular communication and coupling, bone cell gene signatures and metabolic profiles.

• PREDICTION VALUES of heart rate variability (Can assessment with Vagus(TM), skin autofluorescence (by AGE Reader from Diagnoptics) and muscle mass (by DXA whole-body scan) and - strength (by hand grip strength measured by dynamometer) Data will be summarized using odds ratios, trend analysis and regression analysis, adjusting for potential confounders. Students t-test and Chi square tests will be applied for test of group comparison. Statistical analyses will be conducted in Excel® and STATA®.

Power Calculation The total number of individuals that will be recruited is based on a previous study of bone strength in T2D patients (same method) showing bone material strength index (BMSi) of 78,2 (SD 7,5) in controls and 74,6 (SD 7,6) in T2D patients. With 111 T1D patients and 37 controls, the power of the study will be 80% to demonstrate a difference in bone strength of 4.

The number of participants needed for the studies on cell metabolism is based on an expected between-group difference in basal respiration of 25% when using the Seahorse extracellular flux analyser. (Difference of 25% is similar to that observed in another cell type in T1D and non-T1D patients in an on-going study at Aarhus University).

source data will be available on the scanners and as for other examinations, screenshots/photographs of result windows are saved in the encoded database, where the results are also typed in by hand.

Local monitoring will be carried out. Standard Operating Procedures for examinations are followed by involved clinicians and investigators.

Recruitment information / eligibility

• Status: Active, not recruiting
• Enrollment: 148
• Est. completion date: April 1, 2024
• Est. primary completion date: April 1, 2024
• Accepts healthy volunteers: Accepts Healthy Volunteers
• Gender: All
• Age group: 18 Years to 80 Years

Eligibility

Inclusion Criteria:

• T1D subjects: Clinical diagnosis of T1D <18 years of age, C-peptide <200 pmol L-1 at any time point.

• Controls: HbA1c <44 mmol mol-1

Exclusion Criteria:

• BMI < 18.5 kg/m2

• Primary hyperparathyroidism and other calcium metabolic conditions

• Paget's disease and other metabolic bone diseases

• Vitamin D <25 nM (re-test after vitamin D replacement acceptable)

• Known disorders affecting bone metabolism, e.g. uncontrolled thyrotoxicosis, chronic kidney disease (eGFR <30 ml min-1), liver dysfunction (alkaline phosphatase higher than twice the upper limit), celiac disease not controlled by diet, known hypogonadism, severe chronic obstructive pulmonary disease, hypopituitarism and Cushing's disease

• Antiresorptive or bone metabolic drugs for the last 12 months

• Use of anabolic steroids in the last 12 months

• History of pancreatitis

• Allergy to the medicines used

• Inability to give informed consent

• Pregnancy

Related Conditions & MeSH terms

• Diabetes Mellitus, Type 1
• Osteoporosis

Locations

Country	Name	City	State
Denmark	Steno Diabetes Center Nordjylland	Aalborg

Sponsors (7)

Lead Sponsor	Collaborator
Odense University Hospital	Aalborg University Hospital, Aarhus University Hospital, Hospital of South West Jutland, Molecular endocrinology department (KMEB), Steno Diabetes Center Nordjylland, Steno Diabetes Center Odense

Country where clinical trial is conducted

Denmark, 

Outcome

Type	Measure	Description	Time frame	Safety issue
Other	Cortical area	measured By High Resolution periphery Quantitative Computed Tomography(HRpQCT)	baseline
Other	Endosteal perimeter	measured By High Resolution periphery Quantitative Computed Tomography(HRpQCT)	baseline
Other	Periosteal perimeter	measured By High Resolution periphery Quantitative Computed Tomography(HRpQCT)	baseline
Other	Bone Stiffness	measured Finite Element Analysis by High Resolution periphery Quantitative Computed Tomography(HRpQCT)	baseline
Other	Trabecular Thickness (Tb.Th)	measured By High Resolution periphery Quantitative Computed Tomography(HRpQCT)	baseline
Other	Trabecular number (Tb.N)	measured By High Resolution periphery Quantitative Computed Tomography(HRpQCT)	baseline
Other	Other structural and remodelling measures from bone histomorphometry	Osteoid surface (OS/BS, %), Osteoblast surface (Ob.S/BS) and Eroded surface (ES/BS, %)	baseline
Primary	Mitochondrial function: oxygen consumption rate (OCR)	using the Seahorse Xfe96 Analyzer and measurement of glucose uptake of MSCs, OBs and OCs	baseline
Primary	measurement of glucose uptake of MSCs, OBs and OCs	using a bioluminescent assay (Glucose-Uptake Glo, Promega)	baseline
Primary	cell lactate production	using the Seahorse Xfe96 Analyzer and measurement of glucose uptake of MSCs, OBs and OCs	baseline
Primary	Bone Material Strength index (BMSi)	measure of microindentation on tibia by OsteoProbe(TM),. Measure given as index: BMSi. The strength of the bone is higher with higher BMSi measures which measures from approx. 40 to 90 (no unit)	baseline
Primary	Bone mineral density by DXA	DXA scan of hip and spine	baseline
Primary	Spine trabecular bone score (TBS)	estimated by software of DXA on spine using Hologic Horizon A. Approx. 1000 to 1600 with higher quality of the network with higher values of TBS	baseline
Primary	Cortical thickness(Ct.Th)	measured By High Resolution periphery Quantitative Computed Tomography(HRpQCT)	baseline
Primary	Failure load	measured Finite Element Analysis by High Resolution periphery Quantitative Computed Tomography(HRpQCT)	baseline
Primary	volumetric Bone material density (vBMD)	measured By High Resolution periphery Quantitative Computed Tomography(HRpQCT)	baseline
Secondary	microRNA and gene network analyses	Gene expression and activity in cell samples from blood and bone marrow by established methods and including analyses of microRNA and gene network analyses	baseline
Secondary	microarchitecture	By ultramicroscopy of histologic bone samples, the microarchitecture can be analyzed and compared in the case and control groups	baseline
Secondary	Mineralizing surface (MS/BS, %)	By analyses of bone histomorphometry in the case and control groups as a measure of the bone remodelling in T1D bones	baseline
Secondary	Mineral apposition rate (MAR micrometer/day)	By analyses of bone histomorphometry in the case and control groups as a measure of the bone remodelling in T1D bones	baseline
Secondary	Bone formation rate (BFR/BS)	By analyses of bone histomorphometry in the case and control groups as a measure of the bone remodelling in T1D bones	baseline
Secondary	bone turnover markers	By analyses of bone bone turnover markers P1NP, CTX and sclerostin and comparison of the the case and control groups we will analyse the bone remodelling in T1D bones	baseline
Secondary	Tb bone volume fraction (BV/TV)	measured By High Resolution periphery Quantitative Computed Tomography(HRpQCT)	baseline
Secondary	Trabecular thickness (Tb.Th)	measured By High Resolution periphery Quantitative Computed Tomography(HRpQCT)	baseline
Secondary	Cortical porosity(Ct.Po)	measured By High Resolution periphery Quantitative Computed Tomography(HRpQCT)	baseline
Secondary	skin autofluorescence (SAF)	measured by AGE Reader(R) as a surrogate marker for bone AGE content. measures from appr. 0.5 to 4.5 (no unit). The higher the value the greater the tissue accumulation of fluorescent advanced glycation end products	baseline
Secondary	Handgrip Strength	Handgrip Strength in kg measured with hydraulic HandGrip Dynamometer as one of two measures of sarcopenia	baseline
Secondary	Lean mass	Estimated lean mass from DXA wholebody scan as one of two measures of sarcopenia	baseline
Secondary	Cardiologic Autonomic Neuropathy measure (CAN)	by Vagus (TM) as a measure of autonomic neuropathy which returns CAN score of 0 or zero in each of three measurements (rise up, deep breathing and valsalva manoeuvre), calculating a total CAN score of 0 to 3 where 0 is no CAN, 1 is borderline CAN and 2 is a positive CAN screening.	baseline
Secondary	Trabecular spacing (Tb.Sp)	measured By High Resolution periphery Quantitative Computed Tomography(HRpQCT)	baseline
Secondary	Trabecular area	measured By High Resolution periphery Quantitative Computed Tomography(HRpQCT)	baseline