Research Article | Published: 30 September 2021
Characterization of innately decellularised micropattern pseudostem of Musa balbisiana - A non-surface functionalized 3D economic biomaterial scaffold
Deepa Narayanan*, Sarita G Bhat & Gaurav Baranwal
3D micropattern scaffold; banana pseudostem scaffold; economical 3D scaffold; hepatocyte cell spheroids; natural cellulosic 3D scaffold
2. Materials and Methods
2.1 Sourcing of the MPM-3Ds material, preservation, sterilization and 3D scaffold incorporated polystyrene plate development
2.2. Determination of the extend of decellularisation of the pseudostem by DNA quantification
2.3. Taxonomic identification of the MPM-3D source plant
2.4. MPM-3Ds Characterisation
2.5. Estimation of elements and biomolecules in the MPM-3Ds
2.6. Cell attachment, proliferation and differentiation studies on the MPM-3Ds
2.7. Scaffold processing for imaging
2.8. Preliminary in vivo evaluation of biocompatibility and biodegradation in Sprague Dawley rats
2.9. Statistical Analysis
All presented values are the average ± standard deviation. Statistical analyses involved mixed-design ANOVA followed by Tukey’s comparison.
3.1. Sourcing of the MPM-3Ds material, preservation, sterilization and 3D scaffold incorporated polystyrene plate development
3.2. Determination of the extend of decellularization of the pseudostem
The DNA content per mg of tissue from freeze-dried and ETO sterilized pseudostem was 5.6ng, which is only about 10 % of 55.29ng DNA derived from the parent plants leaf. Hence reveals an extended decellularised nature (90%) of the pseudostem [42, 43]. A faint band in agarose gel corresponding to the DNA load in freeze-dried and raw pseudostem (figure 1; lane 1 and 4), when compared to the significant bands corresponding to the DNA isolated from leaf of the offshoot plant and mother plant (figure 1; lane 2 and 3), substantiates our DNA quantification data.
Figure 1: The figure shows the DNA band visualized on the agarose gel from the leaf of the banana mother plant, the offshoot plant form prominent bands when compared to the raw and freeze-dried pseudostem.
3.3. Taxonomic identification of the MPM-3D source plant
The results are furnished in the supplementary results section 2.2 (Suppl. Figure S2, S3).
3.4 MPM-3Ds Characterization
Figure 2: (A) Photograph of the banana pseudostem scaffold with the micro pattern rectangular sockets; (B) the MPM-3D scaffold after 50 days in media showing scaffold integrity; (C) SEM image of the micro patterns evident on the banana pseudostem scaffold; (D and E) SEM image of the interconnecting septa present between the micro rectangular cavities of the MPM-3Ds.
The details are provided under supplementary results section 2.3.3.
3.5. Estimation of elemental composition and biomolecules in the MPM-3Ds
Figure 3: The graphs represent the elemental composition of MPM-3Ds material. (A) The graph shows the mg/L concentration of micronutrients in the sample; the (B) graph shows the mg/L concentration of macronutrient; (C) graph represents the mass fraction weight percentage of CHNS content in the MPM-3Ds material.
Figure 4: The figure shows the chromatogram of the HPLC analysis of the amino acid content in the MPM-3Ds the list of amino acid corresponding to the major peaks obtained in the chromatogram.
3.6. Biocompatibility, cell attachment, and proliferation
Figure 5: (A-E) are the SEM images of the hMSC cultured on the MPM-3Ds and (F) is the calcein stained hMSC on the scaffold on day 14.
Figure 6: (A and B) show the light microscope images of the spheroids formed within the MPM -3D scaffold after the hMSC differentiation treatment, (C and D) show the spheroids that were released into the medium from the MPM-3Ds, and (E) is the calcein stained image of the cells on the scaffold after differentiation.
Figure 7. All the panels depict the different SEM magnification images of the MPM-3Ds bearing cells after hMSC to hepatocyte differentiation. The cells seen here are in the process of aggregating into spheroids.
Figure 8: (A and D) are the bright-field images of a spheroid and OCHIE5 positive red fluorescence emitting; (B) single spheroid; (C) spheroid cluster in the scaffold depth. Cyp2b6 positive green fluorescence emitting; (E) single spheroid and (F) spheroid cluster in the scaffold depth.
Figure 9: The images (A–E) show the different SEM magnification images of mouse cortical neurons on day 14 on the MPM-3Ds.
Figure 10: (A and B) are the H and E stained bright-field images of the subcutaneously implanted MPM-3Ds in male Sprague Dawley rats after one week and four weeks, respectively. The arrows in image (A) point out the MPM-3Ds in the underlying region of the skin after one week of implantation. (C) the graph represents the percentage of polymorph increase in the blood, and (D) is the graphical representation of the C-reactive proteins in mg/mL in the blood of the animal post-implantation when compared to sham-operated and unoperated.
This study has highlighted the MPM-3Ds primary cell, cell line, and differentiation support potential without the need for surface functionalization. The scaffold was not subject to decellularisation and had an extended in vitro integrity, which proves that our selection of the biomaterial candidature of the pseudostem of bananas indeed the best choice of plant tissue for 3D scaffold development. The study has its limitations and requires detailed research on the molecular expressions of differentiated cells with histological evaluation of the in vitro spheroids and a detailed evaluation of its metabolic pathways. Experimental design and implementation for differentiation of hMSC to other cell lineages will add luster to the valuable results obtained here. The physical features of the MPM-3Ds such as its thermal stability up to 100°C and tensile strength of 20.5 ± 2.42MPa with the ability to withstand 228.2 ± 22.75 N force opens up future research on MPM-3Ds and polymer/ceramic/metal-based fusion scaffolding. The development of MPM-3Ds embedded polystyrene cell culture commodity and the critical findings in this study is at the crossroad of immense possibilities for 3D scaffold-based biomedical research and product development.
Dr. Shyamkumar for statistical advice, Centre for Neuroscience CUSAT for donating the mouse cortical neuronal cells.
This research was funded by UGC-DSK Postdoctoral Fellowship, India, grant number: F.4-2/2006(BSR)/BL/16-17/0048.
"The authors have no conflict of interest". "The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results".
Deepa Narayanan & Sarita G Bhat
Department of Biotechnology, Cochin University of Science and Technology, Ernakulum-682022, Kerala, India.
Department of Medical Physiology, College of Medicine, Texas A&M University; 2403(C) Bryan, TX 77807.
Cite this article
Narayanan D, Bhat SG, Baranwal G (2021). Characterization of innately decellularised micropattern pseudostem of Musa balbisiana - A non-surface functionalized 3D economic biomaterial scaffold. T. Appl. Biol. Chem. J; 2(3):76-88. https://doi.org/10.52679/tabcj.2021.0013
Received Revised Accepted Published
05 June 2021 25 August 2021 30 August 2021 30 September 2021