the Type ONe dIabetic Bone Collaboration Study

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.