Division of Biomedical Engineering

University of Saskatchewan

BIOE 990 Seminars

May 3, 2010 (Monday). 1B79 Engineering Building

8:30-8:40am Opening: Dr. FangXiang Wu, Graduate Chair

Session 1: Chair: Mr. Ning Cao

8:40-9:05am: “Charge state determination of peptide tandem mass spectra,” Jinhong Shi,
Ph.D. Candidate.
9:05-9:30am: “FUNCTIONALIZATION OF NANODIAMOND USING CHITOSAN
DERIVATIVES AND PROTEIN,” Hai-Dong Wang, Ph.D. Candidate.
9:30-9:55am: “Characterization of Alginate Scaffold using X-ray Imaging Techniques,”
Guan, Yijing, M. Sc. Candidate.
9:55-10:20am: “Cellulose Nanocomposites- A potential biomaterial for various biomedical
applications,” Sanjai Gopalan, M. Sc. Candidate.

10:20-10:40am: Coffee break

Session 2: Chair: Mrs. Jinhong Shi

10:40:-11:05am: “Development of Cell Damage Law for Biofabrication Processes” Xiaoyu

Tian, Ph.D. Candidate.
11:05-11:30am: “Study on the Effect of Enzymolysis on Chitosan Degradation in Dynamic
Thermal Condition,” Li Zhou, M. Sc. Candidate.
11:30-11:55am: “Creation of Chitosan Microparticles using Ionotropic Gelation Method,”
Shazia Khan, M. Sc. Candidate.

12:00-1:00pm: Lunch
 
Note: Each speaker will have about 20 minutes for presentation and 5 minutes for questions and
discussion. Please upload your slides before 8:30am. The seminar room will be opened about
8:00am.
 Abstracts

Charge state determination of peptide tandem mass spectra

By Jinhong Shi

Tandem mass spectra are the data used to identify peptides. One popular method for peptide
identification with tandem mass spectra is database search. In this method, the experimental
spectra are compared with theoretical spectra which are generated in silico (by computer) from
peptides in a database. For each experimental spectrum, candidate peptides with masses close to
the mass of this spectrum are selected from the database. Then, the search space for this
spectrum can be reduced to the theoretical spectra of these candidate peptides. However, one
problem arises here. Mass spectrometers (equipments which produce mass spectra) can detect a
peptide only if it is charged, and the recorded data is the mass-over-charge (m/z) value of this
peptide ion. We don't know the charge state (z) of the ion, so the real mass of the experimental
spectrum of this peptide is not known to us. And thus, we don't know which peptides are the real
candidates for this spectrum. Current strategies assume each possible charge state (usually +1, +2
and +3) for all spectra and search the database multiple times for each spectrum. Obvious
disadvantages of such kind of methods include
• It is a waste of time to search a database multiple times for each spectrum under the fact
that this spectrum only carries one specific number of charges. This becomes a serious
problem especially when the scale of mass spectra is growing larger and larger.
• It is prone to produce more false positive peptide identifications with current strategies.
To address the above problem, we need to determine the charge states of tandem mass spectra
before the database search. The data we have are tandem mass spectra with many peaks, each of
which has m=z value as x-coordinate and an intensity as y-coordinate. Research work shows that
singly charged (+1) spectra can be 100% correctly determined hierarchically, so the charge state
determination problem can also be formulated as a binary classification problem of doubly (+2)
and triply (+3) charged spectra. In this case, we can use machine learning method, here, we use
Gaussian mixture model (GMM) to classify multiply charged spectra into +2 and +3 peptide ions.

This presentation will introduce how to select new and significantly discriminant features to
describe tandem mass spectra and then use GMM to determine the charge states of tandem mass
spectra.

FUNCTIONALIZATION OF NANODIAMOND USING CHITOSAN DERIVATIVES

AND PROTEIN

By Hai-Dong Wang

At first, the functionalization of nanodiamond particles (ND) for protein drug release was
investigated. N,O-carboxymethyl chitosan (CMCS) was employed to modify the surface
properties of pristine ND particles which has been proved to be successful by a series of
characterizations: Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy, Zeta-
potential measurement, and X-ray diffraction (XRD). The BSA release properties of CMCS
modified nanodiamond particles (NDCMCS) were also studied.

Then the conjugation of bovine serum albumin (BSA) to ND was performed to investigate the
interaction between ND and BSA in ND-BSA conjugate. The conformational change of BSA
upon conjugating with ND was studied by UV-Vis spectroscopy, Fourier transform infrared
spectroscopy, and fluorescence spectroscopy, respectively. The spectroscopic study reveals that
BSA in ND-BSA conjugate undergoes significant conformational change in both secondary and
tertiary levels. In addition, BSA adsorption isotherms and zeta-potential measurement were
employed to investigate the pH dependence of ND-BSA interaction. The adsorption isotherms
obtained at different pHs (3.5, 4.7, 6.0, 7.4, 9.0) and the change in surface charge of ND-BSA
conjugate with pH variations reveal that the conjugation of BSA to ND might not only be
adsorption of BSA onto ND surface but also involves the breaking-up of ND-BSA conjugates by
strong ND-BSA interaction. The results have proven that ND is an excellent platform for the
protein immobilization with very high affinity and this property has great potentials to be widely
applied to the field of biosensor.

Characterization of Alginate Scaffold using X-ray Imaging Techniques

By Yijing Guan

Alginate is a popular biomaterial in tissue engineering, due to its merits of biocompatibility

and biodegradability. When crosslinked with Calcium iron (Ca2+), alginate forms a hydrogel
which provides necessary mechanical support as a scaffold. In this thesis, the material
properties of freeze-dried alginate scaffolds were evaluated using novel micro-computed
tomography (micro-CT) technique as comparison to traditional method such as Scanning
Electron Microscopy (SEM). Micro-CT is a non-destructive and non-invasive X-ray imaging
technique, and requires no segmentation of samples. Another merit of micro-CT is that it can
provide three-dimensional information and enables characterization of alginate scaffold both
qualitatively and quantitative. Important descriptive parameters such as pore size, porosity,
interconnectivity and wall thickness were evaluated. Alginate scaffolds made with different
sodium alginate concentrations and frozen to different temperatures were scanned and
analyzed in micro-CT. Results indicated that lower freezing temperature and higher sodium
alginate concentration lead to smaller pore size, while lower freezing temperature and lower
sodium alginate concentration result in higher porosity.

Cell culture experiments to study the interactions of alginate hydrogel with cells were also
carried out. Schwann cell line was either blended with alginate solution before crosslink with
Calcium Chloride (CaCl2) or put around alginate gel in the culture dish. Light microscopy of
sectioned slices showed that cells surrounded the alginate gel could not grow into the gel, while
cells blended with alginate solution before crosslinking could proliferate inside the hydrogel.
Cells grew inside thin slice of alginate gels appeared to be in better condition and were larger in
size and also grew in clusters. In order to image soft tissue buried inside alginate gels, such as
brain slices, or even cells, novel imaging methods based on Synchrotron Radiation facility were
applied, such as absorption and phase contrast imaging, Diffraction-enhanced imaging (DEI) and
also combined with computed tomography (CT). Synchrotron-based monochromatic x-ray
imaging proved to be good at distinguish objects of similar density, especially biological soft
tissue samples, even without any staining material, such as Osmium Tetroxide (OsO4).

Cellulose Nanocomposites- A potential biomaterial for various biomedical applications

By Sanjai Gopalan,

The term “nanotechnology” have now influenced all major areas such as automobile, food,
pharmaceutical and now the Biomedical Industry too. Due to these developments the biomedical
industry now have a major interest towards cellulosic micro fibril as a component in
nanocomposites, due to its wide abundance, renewable nature, good mechanical property and
biodegradability. Although many researches have been carried out to identify its properties and
advantages, the extraction of cellulose from various sources of plants and microorganisms still
remains as a challenge. The cellulose extracted from bacterial sources along with hydroxyapatite
as reinforcement was found to have a very good property and could be used as a bone material.
The cellulose nanocomposites from plant fibers also had very good properties, which makes it
useful for various biomedical applications based on the reinforcements used. Hence this review
would give a brief idea on nature of cellulose, its extraction, its properties, development and
characterization of a cellulose nanocomposite and its use in the current biomedical industry.

Development of Cell Damage Law for Biofabrication Processes

By Xiaoyu Tian

Novel biofabrication technologies have been explored in recent years, which incorporate live
cells into various processes and systems to manufacture various bio-products, such as tissue
engineering scaffolds, lab-on-a-chip, bio-sensors, and micro-bio-fluidic devices. In these
processes, the cells are subjected to mechanical forces such as hydrostatic pressure and shear
stress. If the forces exceed certain thresholds or shearing time is long enough, cells may be
harmed and even damaged. Beside these two factor, under mechanical force, temperature effect
on cell damage should not be ignored because different temperature can cause the variation of
the mechanical properties of cell membrane and cytoskeleton, leading to different cell viability
under the same mechanical force conditions.

In order to investigate the influence of shear stress, shearing time and temperature on cell
damage, a series experiments have been conducted. A cone- and -plate rheometer was used as
the force generator and time controller and a water bath was used for temperature control. Cell
damage under different conditions were obtained through experiments, based on which, various
mathematical models was employed to represent the cell damage under these conditions. These
mathematical model serve as cell damage law and will be applied in biofabrication process to
predict cell damage and even optimize biofabrication process to minimize cell damage in the
fabrication process.

Study on the Effect of Enzymolysis on Chitosan Degradation in Dynamic Thermal

Condition

By Li Zhou

This research involves in the study of thermal control method for biodegradation of chitosan
scaffolds in tissue engineering, which is the physical device for cells culture and growth. In the
tissue regeneration process, the chitosan scaffold will graduate degrade. What’s more, it is
proved that the enzyme could affect the degradation rate of scaffold significantly.

Thus, my research focuses on developing thermal controllable stimuli, by using enzyme, on the
scaffold so that the degradation can be interfered. Based on such idea, this research would
follows such step: (1) prove that the temperature itself has little effect on degradation rate of
chitosan scaffold; (2) prove that the temperature could affect the activity of enzyme significantly
and get the curve to represent the relation between the temperature and the enzyme’ activity; (3)
design experiment to study the interaction among temperature, activity of enzyme and
degradation rate of scaffold rate.

Further work

The further work of my research will be to study the way to induce temperature change on the
scaffold in vivo. This objective is under the assumption that there is an adequate thermal effect to
control degradation of the scaffold in the human body environment. The general idea to achieve
this objective is to design in-situ carbon nanotube sensor and heater system. The sensor will
measure the temperature of the scaffold and the heater system will give the heat energy if needed.

The proposed research may create ground-breaking result if it is successful. It may lead to
paradigm shift in controlling degradation of scaffolds in the human body environment.

Creation of Chitosan Microparticles using Ionotropic Gelation Method

By Shazia Khan

Ionotropic gelation has been cited as an efficient and biocompatible technique for the preparation
of chitosan microparticles. The method exploits the ionic charge disparity between chitosan and
tripolyphosphate (TPP) inducing electrostatic forces and ionic hardening of the surface of
chitosan particles. Glycosaminoglycans (GAG), such as hyaluronic acid (HA), are naturally
occurring polysaccharides existing within the human body and chitosan, having similar structural
and biochemical characteristics, is considered to be a GAG-analog and therefore biocompatible
and non-toxic to the human body. Over the preceding month, the method of ionotropic gelation
was used in an attempt to create chitosan/HA microparticles.

Chitosan/HA microparticles were initially attempted with the addition of dilute TPP (0.66
mg/mL) to polysaccharide solution (2 mg/mL) i containing Bovine Serum Albumin (BSA) (100
mg/mL)ii under constant stirring (~100 rpm) followed by sonication (1 min). The chitosan/HA
and TPP concentrations, as well as sonication time were systematically varied in an attempt to
increase particle size and formation. It was noted that HA solution would congeal on contact
with the chitosan solution and therefore HA was removed from the procedure. It was difficult to
detect microparticle formation possibly due to the size of the expected particles (300-450 nm)
which was below the limit of detection using light microscopy. A different slightly modified
procedure was usediii by adding dilute (2.5% and later 0.5%) chitosan solution to a concentrated
solution of TPP (10%) using a syringe under constant sonication. The resulting solution
appeared to contain a high number of very fine semi-spherical chitosan particles embedded
within a mesh of fibrous chitosan. Centrifuge filtration was attempted to be used to isolate the
microparticles but this was difficult to accomplish due to particle entrapment within the bed of
highly fibrous chitosan as well as the unknown size range due to modification of the procedure.

From the above experimental methods it was determined that non-uniform microparticles were
created which were very difficult to isolate and therefore future research will involve an alternate
method for microsphere preparation. The water-in-oil (w/o) emulsion technique is also regarded
as a very efficient and biocompatible method for chitosan microsphere preparation. This method
easily allows for control of particle size by varying the size of droplet formation and also
provides simple isolation via filtration and washing with alcohol.iv

                                                            
i
Wadha, S., Paliwal, R., Paliwal, SR., Vyas, SP. “Hyaluronic acid modified chitosan nanoparticles for
effective management of glaucoma: development, characterization, and evaluation” Journal of Drug
Targeting. (2009): 1-11, Early Online
ii
Zhang, HL., Wu, SH., Tao, Y., Zang, LQ., Su, ZQ. “Preparation and Characterization of Water-Soluble
Chitosan Nanoparticles as Protein Delivery System.” Journal of Nanomaterials. (2009): 5 pages
iii
Bodmeier, R., Paeratakul, O. “Spherical Agglomerates of Water-Insoluble Drugs.” Journal of
Pharmaceutical Scinences. 78 (1989): 964-967.
iv
Agnihotri, SA., Mallikarjuna, NN., Aminabhavi, TM. “Recent Advances on Chitosan-based micro- and
nanoparticles in Drug Delivery.” Journal of Controlled Release. 100(2004): 5-28.