Please write a summary of the group comments (1 paragraph) regarding your presentation. This summary should be posted here as soon as possible after the meeting. Most importantly the summary should include key points that you need to address or resolve. I will review your summary at the next meeting and you should be prepared to discuss your progress on these issues.

Meeting Date: July 14, 2009

Attendance:

Excused: Ben. Meeting was in 507.

Meeting Date: July 7, 2009

Attendance: Bin, Wenqian, Daniela, Sandeep, Seda, Javier, Joe, Majid, Halil, Shilpa, Changmo, Laura, Cheong Hoon, Jason, Sherif

Excused: Ben. Meeting was in 507.

General - to use a projector or not? Will try to reserve for next meeting to test. Positives - better images, easier for all to see. Negatives - making presentations too formal.

New members - Laura - UROP working with Shilpa.

Cheong Hoon - new postdoc with Chem Eng background in molecular modeling.

Sandeep presented progress in using GelMA for tissue engineering applications. Results presented included encapsulation studies of EBs in GelMA for 7 days, where there was extensive sprouting, most of which were CD31 positive demonstrating endothelial lineage, although it was difficult to determine whether the EBs were truly in or on the gels (but seperate 2D studies behavior was far different and CD31 negative). Studies underway to investigate using 2 step gel process similar to Celine to ensure EBs encapsulated.

Next 2 weeks - new synthesis with wider range of degree of acrylation, mechanical testing, surface adhesion and spreading in 3D for HUVEC, further analysis of EBs in GelMA - confocal, immuno

Questions were raised what the gel thickness was and whether it or the GelMA % could be increased to ensure encapsulation. Higher % has been tried but is too translucent to effectively image.

Javier presented progress in project on biomasonry. Manuscript preparation is nearing completion with final edits and studies to be completed in the next 2 weeks.

Next 2 weeks - creation of constructs using different shaped microgels, thinner/smaller gels that are multilayer, as well as lock and key, increased cell concentration to at least 1-10 million/ml

Questions were raised as to the use of PEG 1000, and the success of gels smaller than 1 mm/side.

Shilpa presented progress in electrospinning PCL-PGS scaffolds. Presented nearly final figures for paper, a compilation of SEM at different kV and % ratio, fiber diameter, mechanical properties (incomplete), HUVEC proliferation (<< than on TCPS). Problems with contamination has put proliferation data into question.

Next 2 weeks - co-culture SMC and EC on scaffolds for final figure, finish mechanical testing and compare to literature values for cured PGS and electrospun PGSA

Changmo presented progress on MS for creating porous alginate using gelatin microbeads. Manuscript is near completion with all figures nearly complete.

Next 2 weeks - finish and submit manuscript.

Meeting Date: June 30, 2009

Attendance: Bin, Daniela, Javier, Ben, Seda, Sandeep, Wenqian, Joe, Halil, Jason

Excused: Changmo, Shilpa, Lianyong. Meeting was in 507.

Bin updated progress on 3D chitosan/gelatin scaffolds with vascular networks created by freeze drying. Smooth muscle cells are seeded in 100 micron channels while endothelial cells are seeded in 300 microns channels on a separate layer which are stacked in 3-5 layers after 3 days culture to make a multilayer structure. Media is 1/2 SMC and 1/2 HUVEC media as previously reported by others. Results were demonstrated at day 7 and 14 with HUVEC attachment and alignment with the channels.

Next 2 weeks - better histology especially to determine layer interconnection.

Questions were raised how to best image layer interconnection. Suggestions were to fix then cut in half prior to paraffin embedding, dissolving half the material, all without squishing.

Wenqian demonstrated progress in 3D osteon tissue engineering using concentric hydrogel rings assembled using the Du-Majid method. Results were demonstrated using PEG, PEG-RGD, PEG-GelMA, PEG-GelMA-RGD in culture for 5 days. Viability was mixed, very good with spreading for PEG-GelMA +/- RGD, but poor & no spreading for PEG and PEG-RGD. Upon closer inspection on day 9, cells were in wells and not encapsulated in gels, casting doubt on results. Also, in attempts to repeat the results, GelMA would precipitate out of solution with PEG (which is unfortunately supported by the literature).

Next 2 weeks - attempt in PEG-RGD-hydroxyapatite using similar experimental setup as before. HA has shown positive effects on bone cells, leading to binding/spreading.

Questions were raised how to get GelMA to mix with PEG, including changing the conditions of the mixing, such as order, quantities, speed, stirring speed etc.

Halil updated progress on microengraving process to create high throughput arrays for microwell cultures. Results were presented for experiments on the effect of surface chemistry, showing that antibodies would bind to poly l lysine or epoxy coatings, but not uncoated slides. A sandwish eliza was performed, demonstrating a detection limit of 50 ng/ml (Eliza limit ~ 5-10 ng/ml) for poly l lysine and 10 microg/ml for epoxy (but epoxy results very preliminary). In general, epoxy coatings suggest more promise as there was much less non-specific binding and background.

Next 2 weeks - printing into wells, further characterization and quantification with epoxy coated slides.

Questions were raised as to the possible wavelengths of detection and how this affects detection limit (due to detector being unable to match secondary wavelength).

Jason briefly presented progress in MMP-PEG cardiac project. A repeat cell experiment was performed, and while cell viability was much improved, pattern creation and deposition onto slides was suboptimal. The MMP-PEG tended to stay within the PDMS mold rather than bound to the glass slide, presumably due to swelling upon gelation and lack of proper coating on the slides.

Next 2 weeks - investigate cell-free characteristics of MMP-PEG polymerization and binding to coated slides to determine whether further cell experiments are warranted.

Meeting Date: June 23, 2009

Attendance: Javier, Seda, Daniela, Bin, Sherif, Sandeep, Wenqian, Lianyong, Joe, Ben, Halil, Jason

Excused: Changmo, Behnam, Mahdokht. Meeting was in 507.

New members, Joe - new postdoc from EE background at UT Austin and extensive experience with nanofab procedures

Daniela - visiting PhD student in BME from University of Minho in Portugal.

Javier updated progress on biomasonry project. Results on cell-laden 3D constructs were performed, with viability problems seemingly resolved by using 2 stage UV process.

Next 2 weeks - repeat process with 100 micron gels to create multilayer constructs. Video for supplementary data.

Sandeep updated progress on GelMA project. Results presented for micropatterning GelMA down to 100 micron scale, cell encapsulation with viability data (little death). Had problem with contamination after 3 days culture, which was seemingly resolved with improved sterile technique. Also encapsulated 150 micron diameter EBs, which are predisposed to follow vascular lineage, and demonstrated extensive sprouting and good viability after 3 days culture with no GF or other stimulation. EBs cultured ON GelMA lead to FB monolayer spreading, 3D lead to sprouting in >75% of cases within 3 days.

Next 2 weeks - more characterization - methacrylation degree, reaction kinetics, mechanical properties, cell viability and spreading. Also exploring PEG-GelMA with Wenqian. Draft outline for EB project w/Dr Sim.

Ben updated progress in MMP-PEG nanoparticle drug delivery project. General problem (from last meeting) - able to create micro & nano patterns but difficult to do so without film between particles. Coating with trichloro()silane removes the film, but not all molds filled in micropattern, none filled for nano pattern.

Next 2 weeks - Continue to try new techniques to make the micro and nano patterns trying new film coating (PVA), collaborate with Javier who is experienced with some of these techniques.

Shilpa updated progress on PGS/PCL electrospinning for TE. Presented results of optimization of fiber properties in relation to PGS:PCL ratio and voltage, figures for the upcoming paper (and solicited advice on how to present the data and in what order), demonstrated >90% viability in live/dead staining, controls forming fibers and beating on TCPS plates, but is difficult to image scaffold fibers to see if they also are doing this.

Next 2 weeks - SEM to demonstrate cell spreading on fibers, mechanical properties, finishing MS.

Questions were raised as to the difference in morphology between fibers and mat structures, what were the controls - was there a pure PCL control, what is the ideal fiber diameter for these applications?

Meeting Date: June 16, 2009

Attendance: Behnam, Sandeep, Ben, Changmo, Javier, Halil, Lianyong, Sherif, Mahdokht, Wenqian, Bin, Jason, Ali

Excused: Seda, Celine, Majid, Shilpa. Meeting took place in Ali's office.

Jason updated progress on MMP-PEG cardiac tissue engineering project. New cardiomyocyte experiments were performed, except all cells died, even control CM plated in flasks. At end of isolation, water bath was discovered to be 50 deg C, most probably causing the cell death.

Next 2 weeks - repeat experiment now that water bath has been fixed.

Lianyong introduced new project "silk based hydrogels with high strength". Silk chosen due to good mechanical properties, biocompatibility, ability to spin into fibers. Actual project is to use silk fibers to reinforce GelMA hydrogels in an IPN type arrangement.

Next 2 weeks - prepare outline on this topic so as to better evaluate the potential and possible results and pitfalls.

Wenqian updated progress in bone osteon tissue engineering. HUVEC showed good viability in PEG-RGD, while images were shown in PEG vs PEG-RGD. Also outline was revised.

Next 2 weeks - cell viability assessment, immuno staining of endothelial markers CD31 and vWF.

Questions were raised whether cells could spread in PEG-RGD when they cannot degrade it. Literature suggests they can, however the mechanism is unclear.

Halil presented outline on microengraving technique to detect liver cell function in microarray system including results with immunodetection.

Next 2 weeks - standard curve of albumin to determine detection limits, creation of epoxy coatings and comparison with poly l lysine coated slides in primary antibody immobilization and background staining.

Questions were raised how this technique was different from others, advantages over eliza assays that are commercially available.

Ali had general comments for all regarding demonstrating progression in your work and experiences vs series of non-progressing preliminary studies.

Meeting Date: June 9, 2009

Attendance: Ben, Javier, Bin, Sandeep, Wenqian, Halil, Seda, Shilpa, Changmo, Behnam, Mahdokht, Jason

Excused: Celine, Majid. Meeting took place in 507, Ali was not present.

Shilpa updated progress on electrospinning PGS with PCL. Viability and spreading along fibers was good on scaffolds and control plates, thickness limited to < 50 microns, optimized voltage conditions presented for each PGS/PCL ratio to create fibrous scaffolds.

Next 2 weeks - work on reproducibility of fiber/scaffold characteristics, finish SEM imaging, experiments for 2:1 and 4:1 ratios, measure the mechanical properties and fiber diameter

Questions were raised as to what are the desired mechanical properties, cell adhesion properties and how to increase the thickness to > 1-200 microns

Ben updated progress on MMP-PEG nanoparticles for drug delivery. Results were presented for 15 micron sized particles, however SEM was difficult to image clearly. Square gels possible on TPSPMA treated slides.

Next 2 weeks - attempt on fluorinated Si wafer to eliminate glass slides and dust, optimize system to get more uniform particle size, synthesize MMP-PEG (all experiments to date created with PEGDA 550

Questions were raised about whether emulsion would be a better technique, why he was using PEGDA 550 and what shapes were achievable.

Javier updated progress in assembly of gels around PDMS molds. Thicker gels (1 mm) make 1 layer coatings, 500 micron gels more likely to make less controllable multilayer structures, and there is difficulty in minimizing holes in the structure. Able to make cylinders, hemispheres and other shapes.

Next 2 weeks - continue to optimize system with smaller gel size, cell-laden gels and minimization of gaps between gels

Questions were raised about how to control cell distribution, potential leakage between gels, control of multilayer structures and block characteristics.

Behnam/Mahdokht are completing their manuscript and aim to have it completed in the next 2 weeks.

Changmo updated progress on gelatin microsphere based pores in alginate. Cell viability was quantified but statistics for the 80% case were not clear, WST-1 mitochondrial activity showed significant differences and varied with gelatin percentage.

Next 2 weeks - reanalyze albumin assay for out of range results, analysis of cell size, further completion of manuscript.

Also, picking back up on soft lithography of PEG using PDMS w/no wafer.

Questions were raised about dilutions of albumin assay, and a controlled way to measure diffusion and pore connectivity

Meeting Date: May 29, 2009

Attendance: Celine, Shilpa, Behnam, Wenqian, Changmo, Ben, Javier, Halil, Seda, Sandeep, Jason

Excused: Du, Bin, Lianyong. Ali was present; the meeting took place in Ali's office

General: Meetings to change to once per week. Format to change to concentrate on mostly presenting what was done the past 2 weeks and what will be done the next 2 weeks. Get Ali's approval for confocal before going (to conserve $$)

Wenqian has created PEG IPNs using gelatin, however the work was too recent for images.

Questions were raised as to characterization/images of the PEG IPN and the progress achieved since last presentation.

Seda presented progress in the cardiac biosensor project using 3T3 cells as a model system. Cells were binding in the channels as desired, however they also were binding on the surface outside the channels.

It was suggested that she use a 2 layer PDMS system on top of the channels that is non-binding and removable following seeding.

Sandeep presented an outline for creating and characterizing Gelatin-methacrylate as a tissue engineering scaffold. Positives of gelatin are its natural cell binding and degradative properties, low cost and wide availability. Plan includes mechanical evaluation, cell spreading/migration/proliferation within the gel, MMP expression, and micropatterning/microfluidic capabilities.

Questions included differentiating this from previous work by others in using this material for TE, and techniques questions MTT vs DNA vs histology for gels, whether cell counting is feasible/reliable in 3D and how to seed cells only in

proposed microchannels if the gelatin is cell binding everywhere.

Celine/Jason are planning a followup experiment to previous weeks' experiment with MMP degradable PEG and cardiomyocytes. Previous experiment demonstrated ability to create cell-laden patterns but viability was variable.

Questions were raised whether/how this project would proceed when Celine leaves within a month, or whether it should be moved over to other material that is easier to work with (like Gelatin).

Halil presented outline (w/Du & Nezam) for a rapid detection system for assessing hepatocyte function using microengraving techniques. Preliminary data/results included attaching primary antibodies to glass slides, binding of fluorescently tagged secondary to the primary antibody and detection with the microscope.

Questions were raised as to what was completed and what was still outstanding, as well as technique questions of quality of creating PEG microwells - Halil was to publish protocol online for others to use

Meeting Date: May 15, 2009

Attendance: Celine, Shilpa, Mahdokht, Behnam, Majid, Wenqian, Du, Changmo, Ben, Javier, Lianyong, Halil, Jason

Excused: Seda, Bin. Ali was not present; the meeting took place in 507

Behnam/Mahdokht have completed their experiments, and are writing the paper. Jason enlisted to help complete the manuscript.

Shilpa presented progress in optimizing fiber collection, rough vs smooth fibers and preliminary studies of cardiomyocytes (CM) seeded on scaffolds. In 8 days of observation, CM spread in 1 d on TCPS, 3-4 d in Matrigel, difficult to determine on scaffold alone, ?? d on matrigel/scaffolds. Beating noticed on day 2 in Mg/scaffolds, difficult to determine in scaffolds alone. Spreading and attachment to fibers was improved with Matrigel seeding. Overall thickness of scaffolds is 50-70 um with 2-7 um fiber diameter.

Questions discussed included the observed differences between rough and smooth fibers (undetermined so far), the possibility of making multilayer structures, potentially with a different, more

robust hydrogel, and better assessment of the degree of fiber alignment and how this affects mechanical properties and cell alignment.

Changmo presented progress in creating porous alginate scaffolds using gelatin beads in the form of a study to determine gelatin degradation time. After 5 d some gelatin remains in pores, with alginate pore size ~200 nm and gelatin pores ~2-300 um.

Questions were raised as to whether retained gelatin was a + or - for cells, and how to remove this faster if this is not a positive. Also whether the diffusivity could be measured over time to

determine if retained gelatin was restricting transport.

Changmo also presented a revived old project using freezing to create porous hydrogels. The hydrogel lattice prevents ice crystal propagation, which prevents rupture of the cell membranes, reducing the main mode of cell death due to freezing. Also, the pores created appear to be in channel structures, making this a potential method for creating vascular like pore structures in cell-laden hydrogels.

Questions raised regarding the viability of cells after the freezing process (depends on cell type), and potential control over pore size/shape/morphology

Ben presented a revised outline for MMP degradable PEG nanoparticles for targeted drug delivery. Molds have been acquired with 50, 100 and 200 x 200 nm geometries.

Questions were raised as to the methods for creating the hydrogels in molds vs precipitation, whether the nanogels would require a hydrophobic shell to contain most anticancer drugs which are

hydrophobic, how to get the gels out of the molds intact, and how to assess the nanoparticle formation (SEM vs AFM).

Ben also presented a new project to prove that HA polymer coatings could protect cells from UV exposure, based on previous work. Live/dead images were presented to demonstrate UV resistance. Experiments exposed cell-laden hydrogels to UV exposure in culture hood for 1 hour.

Questions were raised about the need to better quantify the live/dead results, as images were not easy to interpret (and quantitative data is needed), whether lack of temperature control could

lead to more cell death, whether the system absorbs or blocks UV exposure and if either of these mechanisms could interfere with crosslinking (block) or cell viability (absorb).