Cellular Bioengineering and Tissue Engineering Laboratory

Low-Intensity Ultrasound in Regenerative Rehabilitation

Damaged or diseased cartilage seldom heals, leading to joint destabilization. Rehabilitative regeneration therapies that merge advances from regenerative medicine with rehabilitative principles have the potential to create new treatment modalities for cartilage injury. While a multitude of restorative process are important for cartilage repair, there is an interest to focus on the synergy between rehabilitative strategies and regenerative responses mediated by endogenous or transplanted stem cells. One such non-invasive mode of rehabilitative conditioning therapy for healing bone fractures, alleviating osteoarthritic discomfort and restoring cartilage repair is pulsed low-intensity-ultrasound at 1.5 MHz (pLIUS).

Differently from all previous studies that employ pLIUS, our novel approach employs continuous LIUS (cLIUS) and we have undertaken a mathematical analysis of the interaction of an acoustic field with cells and tissues and identified a “cell resonance frequency” at which LIUS induced bioeffects were maximized (see Figure 1). My current research focus is in the in vivo translation of the promising in-vitro results with cLIUS for an eventual clinical application. In pursuit of a clinical application of our promising findings, first, my laboratory is undertaking experiments to understand mechanotransductive cascades that dictate MSC responses under cLIUS that will determine the success of regenerative medicine approaches employing LIUS. We employ RNA-SEQ strategies, miRNA profiling methods and metabolic profile of MSCs exposed to cLIUS to gain a fuller understanding of lineage-specific differentiations of MSCs under cLIUS.

Translation of promising in-vitro findings to an in-vivo situation necessitates an understating of the propagation of LIUS in the joint space as well as to ascertain the ability of cLIUS to promote cartilage repair in a pro-inflammatory environment that is caused by cartilage damage or the operating procedure itself. Limited research has been done to determine the extent of US propagation through the joint space.Hence, we are undertaking the modeling of LIUS propagation in knee joints coupled with an experimental verification of the attenuation, so that optimal transducer placement and LIUS regimens can be identified for an eventual clinical application. In parallel, we are also evaluating the chondroprotective effects of cLIUS using established models of chondral injury.

Figure 1: A) Expression of early response genes after exposure to US. B) Total mechanical energy density in cytoplasm and nucleus for cells attached to a plane in an US field. C) Power signal of a 1.5 MHz, 1kHz repeat wave with initial pressure amplitude of 14,000 Pa.

Notable contributions from my laboratory to the field of bioeffects of LIUS are:

Sarayu Bhogoju, Shahid Khan, and Anuradha Subramanian. 2022. "Continuous Low-Intensity Ultrasound Preserves Chondrogenesis of Mesenchymal Stromal Cells in the Presence of Cytokines by Inhibiting NFκB Activation" Biomolecules 12, no. 3: 434. https://doi.org/10.3390/biom12030434
Neety Sahu, April Miller, Hendrik J. Viljoen, and Anuradha Subramanian.Tissue Engineering Part A. Nov 2019. 1538-1549. http://doi.org/10.1089/ten.tea.2018.0355
Budhiraja, G., Sahu, N. and Subramanian A. Low-intensity ultrasound upregulates the expression of cyclin-D1 and promotes cellular proliferation in human mesenchymal stem cells. Biotechnology Journal, 2017 (10.1002/biot.201700382)
Miller, A. Subramanian, A, Viljoen, HJ. 2017. Frequency sensitive mechanism in low-intensity ultrasound enhanced bioeffects. PLoS ONE. Aug 2017 https://doi.org/10.1371/journal.pone.0181717
Miller, A. Subramanian, A, Viljoen, HJ. 2017. Theoretically Proposed Optimal Frequency for Ultrasound Induced Cartilage Restoration. Theoretical Biology and Medical Modeling. Oct 2017
Miller, A. Subramanian, A, Viljoen, HJ. 2017. A nonlinear model of cell interaction with an acoustic field. J. Biomechanics. May 3;56:83-88. doi: 10.1016/j.jbiomech.2017.03.007
Guha Thakurta S, Sahu N, Budhiraja G, Miller A, Akert L, Viljoen, H and Subramanian A. Long-term culture of human mesenchymal stem cell-seeded constructs under ultrasound stimulation: evaluation of chondrogenesis. Biomed. Phys. Eng. Express 2 (2016) 055016. http://dx.doi.org/10.1088/2057-1976/2/5/055016
Guha Thakurta S., Budhiraja, G., and Subramanian, A. Growth factor and ultrasound-assisted bioreactor synergism for human mesenchymal stem cell chondrogenesis, Journal of Tissue Engineering, 2015, DOI. 10.1177/2041731414566529
Guha Thakurta S., Kraft. M. Viljoen, H.J. and Subramanian, A. Enhanced depth-independent chondrocyte proliferation and phenotype maintenance in an ultrasound bioreactor and an assessment of ultrasound dampening in the scaffold. Acta Biomaterialia, DOI: 10.1016/j.actbio.2014.07.013
Louw, T., Budhiraja, G., Viljoen, H.J., and Subramanian, A. Mechanotransduction is frequency dependent under the cavitation threshold. Ultrasound Med Biol. 2012 Oct;38(10):1734-43
Whitney, N. P., Lamb, A.C., Louw, T. and Subramanian, A. Integrin-Mediated Mechanotransduction Pathway Of Low-Intensity Continuous Ultrasound In Human Chondrocytes. Ultrasound in Med. & Biol., pp. 1–10, 2012.http://dx.doi.org/10.1016/j.ultrasmedbio.2012.06.002
Noriega, S.E., Budhiraja, G. and Subramanian, A. Remodeling of chromatin under low intensity diffuse ultrasound. International Journal of Biochemistry & Cell Biology 44 (2012) 1331– 1336
Subramanian, A., Turner, J. A., Budhiraja, G., Guha Thakurta, S., Whitney, N.P. and Nudurupati, S. S. Ultrasonic Bioreactor as a Platform for Studying Cellular Response to Ultrasound. Tissue Engineering. 2012. DOI: 10.1089/ten.tec.2012.0199

BIO-SEPARATIONS

I began my research career with research forays in the topical area of bioseparations, which is central to both bioprocessing and biopharmaceutical operations. My doctoral work was focused on understanding chromatographic separations based on monoclonal antibodies using both theoretical modeling coupled with experimental validation. I further extended my research in the area of bioseparations by combining material development and theoretical modeling of biomolecule transport in these novel chromatographic materials. Funding provided by National Science Foundation enabled the rational synthesis of porous zirconia particles with hierarchical pore size and pore architecture that can be used in the preparative chromatography of biomolecules (Figure 2). As shown in Fig.2, prior art that uses the polymer-induced-colloidal-aggregation (PICA) method produces 3 to 10 micron particles with pore size of 22 nm, from a 100 nm colloid suspension. An analysis of the mass transport fluxes in zirconia particles with a pore size of 22 nm suggests that pore diffusion is the rate-limiting transport mechanism in the EDTPA-modified zirconia particles. The next logical step was then to produce zirconia supports with particle diameters in the range of 50 to 200 μm and with pore sizes in the range of 35 to 100 nm. We have successfully combined the PICA process and the oil emulsion method (OE) to produce macro- and giga-porous zirconia supports (50-250 microns) (see Figure 2). The pore and throat size distributions showed narrow bi-modal distributions over two distinct size scales: 10-100 nm and 600-3000 nm, respectively. I am interested in exploring the use of these zirconia particles as a green catalyst in 5-HMF formations.

Figure 2: Production of bi-modal macroporous zirconia particles. Colloidal zirconia was processed by PICA and oil-emulsion (OE). Particles were analyzed by SEM and N2 and mercury-intrusion porosimetry. Pore architecture was modeled using fractal analysis.

Notable contributions from my laboratory to the field of bio-separations are:

Subramanian, A. and Kaligotla, H.K. An Analysis of Mass Transport Fluxes In Titania-Based Mesoporous Ceramic Matrices. Powder Technology. 2012. DOI/10.1016/j.powtec.2012.08.007
Pattanayak, A. and Subramanian, A. Production of Meso- and Giga-Porous Zirconia Particles - An Improved Multi-Step Particle Aggregation Process. Powder Technology 192 (2009) 359-366
Subramanian, Anu; Martinez, Blanca; Holm, Jill; Carr, Peter; McNeff, Clayton. A Comparative Study of Monoclonal Antibodies (Mabs) Purified from Cell Culture Supernatant on EDTPA-Modified Zirconia Beads and Protein A-Hyper D Support. Journal of Liquid Chromatography & Related Technologies (2006), 29(4), 471-484
Subramanian, Anuradha; Rau, Anand V.; Kaligotla, Hemanth. Surface modification of chitosan for selective surface-protein interaction. Carbohydrate Polymers (2006), 66(3), 321-332
Sarkar, S. and Subramanian, A. Identification of the mass transfer mechanisms involved in the transport og hIgG in ETPA-modified zirconia. Journal of Chromatography, B: Analytical Technologies in the Biomedical and Life Sciences (2005), 821(1), 124-131
Sarkar, S. and Subramanian, A. Modeling of immunoglobulin uptake by N,N,N',N'-ethylenediaminetetramethylenephosphonic acid-modified zirconia particles under static and dynamic conditions. Journal of Chromatography, B: Analytical Technologies in the Biomedical and Life Sciences (2005), 821(1), 81-87
Subramanian, A., Mascoli, C., Roy, S. K., and Hommerding, J., "The Use of Modified Chitosan Macrospheres in the Selective Removal of Immunoglobulins," Journal of Liquid Chromatography, 24(17) (2004) 2649-2670

FUNCTIONAL BIO-MATERIALS

I anticipate that my research efforts in the area of bioseparations will provide the fundamental understanding and basis for the development of surfaces with hierarchical structures and embedded biorecognition at interfaces. Briefly, surfaces that were derivatized with peptides, having albumin binding affinities, were noted to passivate the surfaces; yielding lower platelet adherence and preserving the normal discoid shape of the platelets under static and shear conditions. We demonstrate that the surface passivity by albumin-binding surfaces is conferred by the ability of surface-bound albumin to either impede the co-adsorption of competing proteins or restrict the hemostatically active conformations of proteins. The real-time protein adsorption kinetics on albumin-binding surfaces was evaluated by dynamic in situ spectroscopic ellipsometry.

Figure 3: Generation of albumin-binding surfaces. Silicon-based surfaces were modified using directional silanes upon surface hydroxylation and capped with albumin-binding peptides. Ellipsometry was used to characterize protein adsorption on these surfaces. Surfaces were exposed to blood in a flow-chamber at different shear rates. Control surfaces has activated platelets whereas albumin-modified surfaces had passivated and discoid platelets.

Notable contributions from my laboratory to the field of bio-compatibility of implantable bio-materials are:

Kador, K. E. and Subramanian, A. Sequential Immobilization of recombinant EPCR and thrombomodulin onto Biomedical Grade Polyurethane. Acta Biomaterialia 7 (2011) 2508–2517
Kador, K. E. and Subramanian, A. Controlled and Sequential Immobilization of Proteins onto Biomedical Grade Polyurethane. (In Press: Journal of Biomaterial Science-Polymer Edition 2010. DOI:10.1163/092050610X529191)
Guha Thakurta S. and Subramanian, A. Fabrication of dense, uniform aminosilane monolayers: A platform for protein or ligand immobilization. Colloids and Surfaces A. 2012. DOI. 10.1016/j.colsurfa.2012.08.049
Guha-Thakurta, S., Miller, R. and Subramanian, A. Adherence of platelets to in situ albumin-binding surfaces under flow conditions: role of surface-adsorbed albumin. Biomed. Mater. 7 (2012) 045007 (10pp) doi:10.1088/1748-6041/7/4/045007
Guha Thakurta, S., Viljoen, H.J. and Subramanian, A. Evaluation of the real-time protein adsorption kinetics on albumin-binding surfaces by dynamic in situ spectroscopic ellipsometry (Thin Films (2011) 10.1016/j.tsf.2011.09.084)

SYNTHETIC GENES

The de novo synthesis of genes is emerging as a powerful tool in biotechnology. The ability to synthesize genes of any desired sequence opens the door to seemingly unlimited research possibilities. Our approach employs rapid gene synthesis by polymerase chain assembly (PCA) in a high speed thermocycler in combination with smart error control strategies to effectively minimize thermal damage. We have used this technology-tool available in our laboratories in the successful de-novo assembly of genes encoding chimeric proteins and gene promoter elements upto 3.8 kilobases (M.J.Schenider, MS Thesis, UNL-Lincoln and Joel E. TerMaat, Ph.D Thesis, UNL-Lincoln, TerMaat et al., 2012). Syntheis of designer enzymes that can incorporate the functional domains and character of separate enzymes (i.e. cellulases, hemicellulases and xylanases) to yield a multi-domain, multi-functional enzyme holds immense promise in biotechnological applications involving conversions for harvesting energy from lignocellulolytic biomass. Further, my laboratory has the expertise to express novel genes in both bacterial and Pichia Pastoris system and further purify them using affinity and other chromatographic methods.

Figure 4: Strategy for de novo gene synthesis of a gene encoding a multimeric protein. Input sequence that encodes the desired domains of multiple proteins is first codon optimized and oligo sets are designed. Using PCA process the oligo’s are stitched and PCR amplified to yield intermediate PCR products, which are then stitched to obtain the final “desired gene”. Using tools of recombinant DNA technology the gene product will be subcloned into appropriate expression systems.

Notable contributions from my laboratory to the field of gene-synthesis and protein expression are:

Louw,T., Subramanian, A., Gomez, O. and Viljoen, H. J Mathematical modelling of functionalized-microsphere based assays for rapid DNA detection: Biochemical Engineering Journal, 2014, V(88), 50-60
TerMaat, J. A., Mamedov T.G., Pienaar, E., Whitney, S.E. and Subramanian, A. Automated two-column purification of Iminobiotin and BrdU labeled PCR products for rapid cloning: application to genes synthesized by Polymerase Chain Assembly. J.Chromatographic Science., Vol. 48, pp 1-5, January 2010
TerMaat, J.R., Pienaar, E., Whitney, S. E., Mamedov, T. G. and Subramanian. A. Gene synthesis by integrated polymerase chain assembly and PCR amplification using a high speed thermocycler. Journal of Microbiological Methods, 2009. doi:10.1016/j.mimet.2009.09.015
Mammedov, T.G., Pienaar, E., Whitney. S. E., TerMaat, J. R., Carvill. G., Goliath. R., ,Subramanian,. A. and Viljoena. H. J. A fundamental study of the PCR amplification of GC-rich DNA templates. Comput Biol Chem (2008), doi:10.1016/j.compbiolchem.2008.07.021

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