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Biomaterials

Biomaterials - Poster Session E

Poster N9 - Designing Bone Marrow Analogs for Modeling Bone Mechanical Environment

Saturday, October 26, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  

Introduction:
Mesenchymal stem cells (MSCs) are useful in bone tissue engineering due to their capacity to differentiate into osteoblasts, the cells responsible for bone formation [1] . Differentiation capacity of MSCs is influenced heavily by their microenvironment [2,3] . Recently, our team has developed Bone Marrow Analogs (BMAs) to study the role of bone architecture on the mechanoregulation of MSCs. These BMAs consists of PLA based trabecular structure and cell-laden, hyaluronic acid-based hydrogels with MMP cleavable and cell attachment sites to mimic bone marrow. Results indicated that reducing trabecular bone volume over total volume (BV/TV) from 25%, representing healthy bone, to 13%, typical of aging bone, significantly reduces collagen-I production under mechanical stimuli. We hypothesize that decreased BV/TV reduces collagen-I production via alteration cellular mechanical environment of MSCs. Shown in Fig.1A, surface features of PLA-based printing show limited accuracy, compromising the repeatability and necessitating the development of more precise trabecular scaffolds. In this way, here we explore the possibility of using better-defined scaffolds printed via stereolithography (SLA) [4] . Specifically, we tested the viability of Biomed Clear Resin (BCR, Formalabs, RS-F2-BMCL-01), which showed improved accuracy (Fig. 1B). To compare the performance of BCR against PLA, we first evaluated the surface properties (Fig. 1B) and cell attachment (Fig. 2A) followed by cell proliferation experiments (Fig. 2B). Finally, following the currently ongoing osteocompatibility experiments, we plan to compare osteogenic differentiation of BCR and PLA based BMAs.

Materials and

Methods:  3D Printing and Ink: A Form 3B+ printer was utilized to fabricate scaffolds using BCR. BCR was chosen for its biocompatibility. It is important to note that PLA scaffolds were printed using the Prusa MK4, as detailed in [4] .
Surface Modification: To improve cellular adhesion, plasma treatments with either Ar+O2 or Ar were employed as follows, 1) Ar+O2, 200s,12.4W, 2) Ar , 200s, 11.4W, 3) Ar +O2, 90s, 12.6W, 4) Ar +O2, 200s, 12.6W, 5) Ar , 200s, 9.6W.
Modification and quantification of surface properties: In our experiment using the Biolin Scientific T200-Auto3, we investigated the wettability of various substrates through sessile drop analysis, determining the contact angle to assess hydrophilicity or hydrophobicity. We quantitatively compared the relative hydrophilicity of different substrates, with lower contact angles indicating higher hydrophilicity.

Quantification of cell attachment: Cellular morphology and cytoskeletal organization were evaluated using Phalloidin-iFluor™ 488 Conjugate (Cayman Chemical) for actin staining. Nuclei were visualized using NucSpot Live 650 (Fisher Scientific) to observe cell distribution Quantification of cell proliferation: Cell proliferation was assessed using the XTT Cell Proliferation Assay kit (Cayman Chemical) to measure metabolic activity of osteoblast-like cells.
Osteogenic differentiation and analysis: We are currently conducting the osteogenesis process to compare disks and scaffolds (in two groups: PLA and BCR) by seeding them in osteogenic media and conducting the differentiation process for 21 days. After 21 days, all samples will be fixed with 4% PFA, stained with Xylenol Orange and NucSpot 650, and imaged using a Zeiss confocal microscope.

Results, Conclusions, and Discussions: 

Results:
Figure 1A shows scaffolds with different porosity (13%, 25%) printed with BCR and PLA. Samples 4 and 5, representing plasma treatments with AR and AR + O2 , were selected for further study due to their smaller contact angles, indicating higher substrate hydrophilicity and better cell attachment (Figure 1B). The fluorescent images in depict phalloidin and Nuc red staining to observe cell proliferation with Sample 4 and Sample 5 using ImageJ for cell counting (Figure 2A). Due to challenges in directly comparing different groups, an XTT assay was performed for comparative efficacy. The XTT assay results indicate that plasma treatment did not affect the proliferation of MSCs. The number of cells in the presence of
BCR without plasma treatment was similar to those with PLA. Therefore, we decided to proceed with the sample without plasma treatment, as the plasma-treated samples did not show improved results (Figure 2B).

Discussion:
This study shows that using BCR in SLA printing significantly advances scaffold creation for bone tissue engineering, enabling precise porosities mimicking healthy and aging bone. Plasma treatments improved initial cell adhesion but did not significantly enhance MSC proliferation or
osteogenesis compared to untreated BCR. The findings underscore the critical role of scaffold architecture in MSC differentiation, justifying the use of untreated BCR for simpler, cost-effective results.

Conclusion:
This study highlights the potential of SLA-printed BCR scaffolds with precise porosities for bone tissue engineering, effectively mimicking age- related trabecular bone structures. While plasma treatments showed no significant impact on MSC proliferation, untreated BCR scaffolds were equally effective. The research underscores the importance of scaffold architecture on MSC behavior, suggesting further optimization to enhance osteogenic outcomes and advancing personalized regenerative therapies for improved patient outcomes.

Acknowledgements (Optional):  References
[1] Y. Gou, Y. Huang, W. Luo, Y. Li, P. Zhao, J. Zhong, X. Dong, M. Guo, A. Li, A. Hao, G. Zhao, Y. Wang, Y. Zhu, H. Zhang, Y. Shi, W. Wagstaff, H.H. Luu, L.L. Shi, R.R. Reid, T.-C. He, J. Fan, Adipose-derived mesenchymal stem cells (MSCs) are a superior cell source for bone tissue engineering, Bioact. Mater. 34 (2024) 51–63. https://doi.org/10.1016/j.bioactmat.2023.12.003.
[2] Q. Gao, J. Liu, M. Wang, X. Liu, Y. Jiang, J. Su, Biomaterials regulates BMSCs differentiation via mechanical microenvironment, Biomater. Adv. 157 (2024) 213738. https://doi.org/10.1016/j.bioadv.2023.213738.
[3] Z. Feng, M. Jin, J. Liang, J. Kang, H. Yang, S. Guo, X. Sun, Insight into the effect of biomaterials on osteogenic differentiation of mesenchymal stem cells: A review from a mitochondrial perspective, Acta Biomater. 164 (2023) 1–14. https://doi.org/10.1016/j.actbio.2023.03.032.
[4] A.M. Regner, M. DeLeon, K.D. Gibbons, S. Howard, D.Q. Nesbitt, T.J. Lujan, C.K. Fitzpatrick, M.C. Farach-Carson, D. Wu, G. Uzer, Increased deformations are dispensable for cell mechanoresponse in engineered bone analogs mimicking aging bone marrow, BioRxiv (2023).