Hyaluronic acid surface properties and single crystals, Rubner's lab
HA molecular weight dependency on physical properties was dramatically determined in solution and in the biological matrix. However, the molecular weight dependency of HA regarding physical properties on surfaces has not been determined. HA interaction with surfaces and its hydrodynamic properties may be exploited for device fabrication. I filed a provisional patent for my NANOBITS technology just to prove I developed the technology only. Without collaborators it would be impossible to pursue this vision. I was pleased that I brought closure to this aspect of my work.
The scaffolding of HA fragments on surfaces through physical manipulation. See enlarged version of this image.
The manipulation of HA fragments using electromagnetic radiation, temperature, etc., during dehydration could lead to a novel way of creating biocompatible electroconductive nanofiber mesh.
The dehydration of hyaluronic acid from aqueous salt solutions could lead to the creation of biocompatible electroconductive matrices for cell studies and novel device fabrication.
In Dr. Rubner's lab I had the opportunity to explore the properties of hyaluronan or hyaluronic acid on various surfaces. This approach is very different from my previous work in aqueous solution and in cell culture. The evaporation of water from the hyaluronan (HA) fragments resulted in an array of interconnecting fibrous networks containing cables and hydrated hubs. The molecular aggregates linked together in heirarchical interlocking patterns one would expect for a scaffolding molecule responsible for tissue matrix genesis. The shape and molecular contours of the HA hexamer and tetramer suggest that HA may have been the precellular incubator for stem cell genesis. The structure I propose for my NANOBITS can serve as an incubator or hub for the human telomeric repeat d[AG3(T2AG3)3] G-tetraplex reported to be the start sequence for DNA proliferation. It was after this discovery that I coined my conjecture "Biomatrixgenesis". Reverse engineering using the principles found from my research will be used by others someday to fabricate biocompatibe electroconductive nanofiber mesh.
Realizing that I had gone as far as the system would allow in mt search for the genesis molecule of the bible, I left MIT with a desire to develop a new strategy to prove my conjecture. This strategy was to use art and Divinity in concert with science. BIAS Incorporated, which I founded in 2002 became the organizational entity for this work.
Future research will be published in my book (manuscript copyrighted 2008-pending),
Micromass culture (below) shows HA hexamer and MabIVd4 inhibition of chrondrogenesis
Chondrocyte nodules after 4 day culturing of chick embryonic limd cells +/- HA and anti-HABP. See enlarged image.
How would HA behave on surfaces with respect to its concentration, molecular chain length, and its degree of hydration? It was this thinking that led to the work completed in the Rubner Group at MIT.
HA mediated cell cell adhesion and cell motility for HABP containing cells. See enlarged image.
HA is prevalent throughout the mammalian tissue matrix. Matrix scaffolding for cell proliferation and tissue engineering involves hyaluronic acid proteoglycans and hyaluronic acid binding proteins. It is highly probable that the initiation step for the process was an HA tetramer or hexamer capable of forming an array of individual nanoscale engines or precellular incubators.
Perhaps the presence of HABP on matrix cells is more than a coincidence. See enlarged image.
It may be that HA hexamers formed nanoscale precellular incubator through self-assembly, which are capable of scaffolding a natural biocompatible electroconductive nanofiber mesh.
The self-assembly of the simple carbohydrate polymer, hyaluronic acid in aqueous salt solution, must have a unique purpose in the scaffolding of cellular life supporting matrices"
In Dr. Tooles lab I had the opportunity to explore the effects of hyaluronan (HA) molecular weight on cell morphogenesis and extracellular matrix engineering. In my dissertation I had rheological evidence that HA fragments reduce the viscosity of HA polymer plus fragment mixtures. We learned that segmental interaction between the extracellular matrix fragments bound to HABP and the larger polymer molecules play a definitive role in tissue morphogenesis and tissue matrix engineering. The question that remained for me was the nature of the relationship between HA, DNA, and protein with regard to directing and mediating the morphogenesis process. It was becoming clear to me that HA was the scaffolding molecule.the MATRIX.
This close-up of HA nanofiber shows the anchoring and overlap of hierarchical segments. The spinning sphere represents the possible binding site of nanoparticle clusters. Such binding of nanoparticles show patterning in TEM images like those seen by SEM and OM, which support the hierarchical patterning of HA.
HA hierarchical crystal binding region
The research done at Tufts University gave me the opportunity to explore the cellular properties of hyaluronic acid (HA). The cellular properties was a natural step in my research from having studied the physical properties of HA under physiological conditions in solution.
Toole BP, Banerjee SD, Turner RE, Munaim S, Knudson CB (1992) HA–cell interactions in limb dev.: In:Dev. Pattern. of the Vert. Limb. Hinchliffe J, Hurle J, Summerbell D (eds.). Plenum. pp.1-9.
Toole BP,Turner RE, Banerjee SD,HA-binding protein in chondrogenesis and angiogenesis in Clinical and Biological Research. 383B:437-44. 1993.
Turner, RE., Banerjee, S.D., Toole, T.P., 1990, Role of HA binding protein in chondrogenesis (unpublished manuscript)
PAGel of hyaluronic acid showing different degrees of polymerization. see enlarged image.
HA was enzymatically digested into various sizes and the sizes characterized using a number of experimental techniques. The interaction of the cationic dye with the HA, a polyelectrolyte, proved to be an excellent screening method for future matrix work.
A proposed model for HA chain uncoupling (See conclusion in Dr. Turner's Ph.D. Dissertation (1986). See enlarged image.
HA was shown to couple and uncouple the HA polymer network based on its solution properties. The model was proposed in my dissertation in 1986. The proposed model held true for cell adhesion and migration. Now it appears to hold for chain-chain interactions on various surfaces.
While self-assembly of DNA and proteins is intriquing, the basic model for matrix self-assembly should be a simple molecule with no strings attached"
In Dr. Cowman's group I was intriqued by the evidence presented at that time that Hyaluronic Acid, a simple carbohydrate polymer, could bind cationic dyes in a chain length dependent manner and show a molecular weight dependency on its physical properties. I spent my entire graduate research experience exploring these solution properties using an impressive array of techniques and methodologies. The potential for self-association by hyaluronate (HA) chains in 0.15 NaCl was investigated, using low molecular weight HA segments as a model system. HA segments were derived from the polymer by controlled enzymatic digestion, and purified by gel filtration chromatography. Seven samples of narrow molecular weight distribution were analyzed by sensitivity-enhanced polyacrylamide gel electrophoresis, and found to have the following weight-average numbers of repeating disaccharide units: A, 90; B, 51; C, 38; D, 31; E, 23; F, 18; G, 13. The segment preparations were studied in 0.15 NaCl by capillary viscometry, low angle laser light scattering, and circular dichroism spectroscopy. The data indicate concentration-dependent intermolecular association of short segments, and a capability for intramolecular association (hairpin formation) by larger HA segments.
After proposing a model from chain-chain interaction for HA in dilute salt solution, the next step was to determine its behavior in actual cell systems. This curiosity ultimately led me to the laboratory of Dr. Toole at Tufts.
My dissertation not only tied for first place for best dissertation at what is now NYU-Poly. I was nominated by the Chemmistry faculty for Signa XI honors in research. This was a definitive work on the physical properties of hyaluronic acid as a function of molecular weight. A search of the literature will show that few around the globe were looking at this problem. Further work on the interaction of individual segments with the matrix as a whole was also explored.
The size dependency of HA in physical properties may be responsible of HA's matrix shape-shifting characteristics. See enlarged version of this image.
Turner, Raymond Edward, Hyaluronic Acid:MW Depend. of Phys. Properties In Dissertation Abstracts, (Ph.D., Polytechnic U.Brooklyn), June 1986.
Turner, R. E, Lin, P.Y., and Cowman, M.K., Self-association of HA segments... Arch. of Biochem. and Biophys. (1988)
Turner, R.E., and Cowman, M.K. Cationic dye binding by HA fragmentsArch. of Biochem. and Biophys. (1985) 237,253 - 260.
