(I-334) An engineered model to elucidate molecular clutch mechanisms of mechanotransduction during bone nanoscale mineralization

Thursday, October 12, 2023

2:00 PM – 3:00 PM PDT

Location: Exhibit Hall - Row I - Poster # 334

Presenting Author(s)

Sofia Vignolo (she/her/hers)

MD-PhD Student
Oregon Health & Science University, Oregon, United States

Co-Author(s)

AA

Avathamsa Athirasala

Post-doctoral fellow
Oregon Health & Science University
Portland, Oregon, United States

Last Author(s)

LB

Luiz Bertassoni, Ph.D.

Associate Professor
Department of Biomedical Engineering, Cancer Early Detection Advanced Research Center, Oregon Health and Science University, United States

Introduction::

Ossification is an important developmental process where bone is created in appositional layers through the gradual calcification of the native extracellular matrix (ECM)¹. This evolution is synchronously orchestrated by biological processes that allow for mesenchymal condensation of stem cells and their differentiation into osteoid-secreting osteoblasts^{2, 3}. While bone mechanobiology has been extensively studied in skeletal development and fracture healing, the interplay between mechanics and biology during the transition of calcifying osteoid to mature bone remains poorly understood⁴. Understanding and replicating bone formation is imperative to support regeneration in critical-sized bone defects which are commonly seen in dental care, orthopedic surgery, trauma causing nonunions/malunions, osteonecrosis, certain osteolytic conditions, and following cancer treatment such as surgical resection⁵.

Our objective is to replicate the critical changes in the ECM during bone formation to study the step-by-step process of bone regeneration in vitro, as current models are limited. Here, we employ a nanoscale materials engineering strategy to recreate the stiffening and nanoscale calcification of bone tissue in a controllable manner using a polymer-induced liquid-precursor (PILP) based strategy for intrafibrillar mineralization⁶. These models enable the unique capability of elucidating the governing role played by the different aspects of the evolving native bone microenvironment such as its composition, nanoscale architecture, and mechanical properties on its biological response. Ultimately, we hope to advance the fundamental understanding of the mechanobiology behind bone formation, contributing to future research on regenerative therapies.

Materials and Methods::

Collagen hydrogels were fabricated in 4 ways to model the different stages of ossification as the bone matrix undergoes stiffening and calcification: 1) soft fibrillar collagen, 2) high density fibrillar collagen (HDFC), 3) low mineralized HDFC and 4) high mineralized HDFC. Soft collagen hydrogels were fabricated on 96-well plates by polymerizing 100 µL of 1.5 mg/mL collagen for 45 minutes at 37˚C. These hydrogels were further centrifuged at 3,500 g at room temperature to allow for compaction into HDFC. The HDFC hydrogels were mineralized using a modified biomimetic calcification medium (α-MEM, osteopontin, CaCl2, K2HPO4) for 1 and 3 days to represent low and high mineralization respectively.

Characterization of the hydrogel model was performed using Alizarin Red to confirm the presence of calcium ion deposition in the mineralized samples, scanning electron microscopy (SEM) to visualize the structural composition, and Fourier-transform infrared spectroscopy (FTIR) to gain information of the chemical bonding/functional groups to quantify the mineral-to-matrix ratio.

After establishing the hydrogel model, bone-marrow derived human mesenchymal stem cells (MSCs) were seeded in 2D with a density of 10,000 cells per well to assess cell response. Samples were fixed at 24 hours to assess the activation of the molecular clutch and its mechanotransducive signaling through immunofluorescent staining for focal adhesion kinase (FAK), phosphorylated paxillin (p-paxillin), Yes-associated protein 1 (YAP-1), and Ras homolog family member A (RhoA). Additionally, live cell imaging with a nuclear and membrane cell mask was performed to understand cell morphology and motility. Image quantification was performed using ImageJ.

Results, Conclusions, and Discussions::

The hydrogel model was thoroughly characterized both qualitatively and quantitatively. Qualitative analysis using Alizarin Red and SEM revealed distinct microarchitecture and mineralization profiles for each stepwise ECM created. SEM showed evident D-banding of collagen in non-mineralized samples while absent in mineralized sample, indicating intrafibrillar mineralization. Extrafibrillar minerals were observed as florets and small protrusions on the fibers of the mineralized samples. The quantitative analysis using FTIR further confirmed mineralization by detecting the presence of the PO4 peak in mineralized samples.

Immunofluorescent staining of FAK and p-paxillin indicated higher expression in mineralized samples evidencing high stiffness sensing, formation of adhesion complexes, and engagement of the molecular clutch under these conditions. However, there was no difference in RhoA activation nor nuclear translocation of YAP-1 between the 4 groups at the 24-hour timepoint. Interestingly, cells exhibited clear differences in morphological response depending on the type of ECM they were cultured on, raising questions about their biological mechanisms in these distinct environments. Given that cell morphology is considered a key indicator of Mechanotransduction, it may be suggested that RhoA and YAP-1 activation may have occurred at an earlier timepoint. These findings are in line with live cell imaging data showing robust adherence and cell motility within one hour of seeding, suggesting that mechanotransducive signaling may be activated rapidly and at very early time points.

In summary, we successfully replicated the stepwise changes in the ECM that occur during bone formation, creating a bone-like environment to mimic the ossification process. We observed differences in cellular behavior in response to sequential stiffening and mineralization in this model which grant the ability to explore further mechanistic questions regarding the cellular response during bone regeneration. The findings of this work contribute to understanding the molecular clutch model of MSCs and shed light on the complex process of bone regeneration.

Acknowledgements (Optional): :

We would like to thank the Knight Cancer Precision Biofabrication Hub and their experienced members for supporting our work. We acknowledge funding from the NIH/National Institute of Dental and Craniofacial Research (R01DE029553 and R21CA263860 to L.E.B) and the Knight Cancer Institute’s Cancer Early Detection Advanced Research Center.

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