Towards a Better Understanding of Osteoporosis: The Role of Solid-State NMR

Bone diseases are on the rise globally, driving the need for earlier diagnosis and better treatment. Osteoporosis is the most common metabolic bone disease, and our aging population is turning it into a global epidemic. With diagnosis difficult until a fracture occurs, there is a push to understand the underlying causes more deeply, to aid early diagnosis of poor bone structure and density and improve options for treatment.

Unlike body fluids or tissue, the fact that bone is a solid has, for a long time, made it difficult to analyze using the usual suite of bioanalytical techniques without destroying the delicate chemical interactions between its constituents.

Now, a team of researchers, led by Dr. Neeraj Sinha at the Centre of Bio-Medical Research (CBMR), Lucknow, India, is using solid-state nuclear magnetic resonance (NMR) to better understand the role of one of bone’s key components – collagen – and show a pathway to improved medical interventions.

Understanding the role of water

Collagen is the most abundant protein in mammals, found in connective tissues, such as bones, cartilage, tendons, ligaments and skin. Its properties depend upon interactions with other components, with water being particularly important.

In human bones, water constitutes about a quarter of the denser outer layer (cortical bone) by volume, and a key area of interest lies in understanding its effect on bone structure. Dr. Sinha and his team designed a series of experiments to investigate how water content affects the bone matrix, a highly dynamic system in which cells undergo continuous regeneration.

Using solid-state NMR, the team could measure the water-dependent collagen–mineral interface, revealing its importance in determining the bone’s mechanical properties. This data gathered on collagen structure, its effect on bone hydration and association with the interface could be used in the design of molecules that bind water into the bone matrix.

Boosting analytical sensitivity

To ensure sharp spectral peaks, solid-state NMR relies upon so-called “magic angle spinning” (MAS). This involves spinning the sample very fast at the “magic angle” of 54.7°, to mimic the tumbling of molecules in solution and suppress line broadening that would otherwise result from interactions involving ¹H nuclei.

Two other methods were also used by the Sinha team to boost sensitivity in the analyses: MAS cryoprobe technology to reduce background noise and dynamic nuclear polarization (DNP) to boost the signal from the atoms of interest – in this case, nitrogen. This gain in sensitivity opened up the possibility of performing advanced experiments to elucidate the 3D structural details in the native environment of bones and cartilage at the natural isotopic abundance.

One of the structural interactions involves citrate, which is abundant in the bone’s extracellular matrix (ECM). Citrate has a high binding affinity to the calcium stored in the hard tissue and is believed to play a key role in regulating metabolic functions to maintain the structural integrity of bone. With the help of DNP solid-state NMR, the CBMR team was able to investigate molecular-level interactions between citrate and collagen inside the bone matrix, and they were able to determine how citrate might influence processes including the collagen-based “templating” of bone mineralization with calcium phosphate (Figure 1).

Figure 1. Natural-abundance 2D NMR spectrum showing the correlation between the signals from ¹³C nuclei (x-axis) and ¹H nuclei (y-axis) in the ECM of bone. The expanded region (1B) shows the molecular interaction between citrate and certain amino acids in collagen, shown by the dotted lines. Credit: Reprinted with permission from N. Tiwari et al., The Journal of Physical Chemistry B, 2021, 125: 4757–4766. doi: 10.1021/acs.jpcb.1c01431. Copyright 2021 American Chemical Society.

The team was able to show that citrate has molecular interactions with the arginine, glutamate and alanine residues, as well as some aromatic amino acids, within the collagen protein. This confirmed that citrate is an integral component of the nanocomposite between calcium phosphate and collagen within the bone matrix.

DNP experiments allowed ¹H−¹⁵N correlation experiments to be carried out using ¹⁵N at its natural abundance – something which has not been possible until now. This provided insights into the backbone and side-chain structural map of the collagen matrix inside the bone.

The minimal sample preparation required was another advantage of the approach. The powdered bone could be analyzed in its natural state, helping the team break new ground in osteoporosis research.

Better diagnosis and treatment of osteoporosis

Future studies at CBMR will look at the application of this research in more detail, to study conditions such as osteoporosis and the bone healing process. These studies will analyze the changes that the onset of osteoporosis brings, as well as the effects of different therapeutic interventions.

A deeper understanding of the mechanism of changes that take place as the bone repairs itself, particularly in the water transfer mechanism, will help inform drug development programs to support and accelerate the bone healing process. In the longer term, a clinical diagnostic method based on measuring water content in vivo may help combat the increasing incidence of osteoporosis worldwide.

About the author:

Prof. Sinha is Dean and Head of the Nuclear Magnetic Resonance/Magnetic Resonance Imaging (NMR/MRI) unit at the Centre of Bio-Medical Research (CBMR). He has worked in the field of NMR spectroscopy for 25 years, starting with his PhD in Physics at the Indian Institute of Science, Bangalore and postdoctorate at the University of California, San Diego, where he used solid-state NMR to research biomolecules and bioproteins using both theoretical and experimental methods. The versatility and accuracy of NMR made it the natural choice to advance his work.