Scaffolds - Science topic

Hi Pavithra,

The submerging method for the incorporation of biomolecules into 2D or 3D scaffolds which is also called "the dip-coating method" is a straightforward strategy, however, one of the inevitable but sometimes negligible problems about this procedure is free unencapsulated/unbound drug molecule residues in the feed solution. The efficiency of this approach is essentially correlated to your final objective.

Wish you the best of luck.

Dear Pavithra Pattabiraman, try extraction. In the following paper it is mentioned how to do that. My Regards

Hi, there are two ways you can determine the presence of any metal substance in a scaffolds.

1. You can do Inductively coupled plasma - optical emission spectrometry, (ICP-OES) or ICP-MS after incubating the samples in specific biological medium for a certain period of time (for leaching study) or after complete degradation.

2. You can also try with EDX-SEM analysis, this also gives you the approximate quantitative results about the presence. Additionally, the distribution of metals inside the scaffold. But this technique has some limitations too, you can try with multiple areas of a same sample to get the average quantitative results.

Hi Simona!

We have the same problem right now. Did you resolve it? if yes, how?

Many thanks!

Hello Matheus.

I am also trying to do the same thing right now and found this question.

Have you succeeded in creating the gel from scaffolds EMC first?

Could you let me know what method you used?

In my case, we want to create a hydrogel from fish testicular scaffolds and then, avaluates the cell colonization.

Thanks!

General considerations without specific PLGA considerations:

With basic treatment, you only cleave ester functionalities, so you shorten your PLGA chains and open functionalities for other reactands.

With acid treatment, both ester cleavage and ester bond formation may happen, so if a random chain length change is desirable for your aspired functionality, acid treatment is your go-to tool.

I should add that to my future experiments. in the mean time, i am limited to protein level only (No primers yet). In my opinion, myotube contraction is a sign of terminal differentiation of myoblasts.

Ravi Kant Upadhyay Which alkali suits best? NaOH or Na2Co3..

You can actually request any experiment demonstration through http://labdemo.unaux.com for better understanding of the protocol.

You will surely have the Carb. met. chit entangled in the GelMa network and probably a fraction will be covalently binded, but I would not rely in that if you want a deep covalent interaction between the polymers. On the other hand, I feel that would be enough to have a solid quite interacting composite.

Thank you Ferry P W Melchels for the suggestions.

You may want to check answers to a somewhat related question at this forum for its possible interest: «What are the most important parameters controlling the extrusion of pastes?»; https://www.researchgate.net/post/What_are_the_most_important_parameters_controlling_the_extrusion_of_pastes

Benjamin Fournier I was considering doing so but I wasn’t sure if cells should/could be lysed straight off the scaffold without detaching first. I am concerned that I won’t extract all of the DNA if cells are trapped on the internal area of the scaffold. I will have to make sure that each internal pore/cavity is exposed to the lysis buffer but I believe it should work. Have you ever tried to lyse cells directly from a scaffold? Thanks!

Samples were fixed by adding 5% glutaraldehyde solution overnight which was replaced with a fresh sterilised solution of PBS and changed three times before soaking in fresh sterilised deionised water for one hour twice. Then, the CCC samples were frozen at −80 °C for three hours before placing them into a Christ ALPHA 2–4 freeze-dryer for 24 hours.

Dear Deniz Sezlev

Yes, you need to know the quantity of RNA before moving to cDNA synthesis and PCR. In my case, I used 200 ng of RNA (except for the sample with 100 ng of RNA) to perform cDNA synthesis, and then I used the samples directly for qPCR without prior dilution. However, for the sample with 100 ng of RNA, I added double the amount of cDNA (and decreased the volume of nuclease-free water in the mastermix).

If you have access to a Nanodrop, it may be helpful to check your samples usgin it

I hope this helps.

Regards,

Farah

Ken Hsu This thread might be useful, have a look https://www.biostars.org/p/9466376/

Hi Archana Rajendran

The attachment of cells on the scaffold will depend on hydrophilicity and surface roughness. You can improve the adhesion of cells on the scaffold by dipping in complete media/FBS before cell seeding or coating it with gelatin.

There are silanes, some of them available in aqueous solution that can be used to coat the dish/well and reduce cell adhesion. They might interfere with adhesion of your scaffold to the dish, however.

there are low cell attachment plates available from many companies used for Organoi/spheroid formation, but they tend to be round-bottomed, not great for some microscopy applications.

You can ask from a local histopathology expert to do it for you or give appropriate protocol for preparation.

, do you have any specific company in mind regarding the biopsy punch? I tried 2 companies and they could not punch through. Have you used onefrom any specific company?

AND IF YOU USE I-PRF, IN A SHORT TIME IT SOLIDIFIES, PROVIDES GROWTH FACTORS AND CELLS WOULD BE MORE "FIRM"

OR PLASMA RICH IN PLATELETS

Dear Luana,

working with cells in scaffold matrices and determining their viability status can be quite challenging. Therefore - for a first (and quite cheap) analysis - I would suggest to simply go for life/dead analysis using the fluorescent dyes DAPI (or Hoechst 33342) and propidium iodide. Diffusion in scaffold materials is no problem (at least in my experiments) and washing to remove unbound dyes can be kept at a minimum since fluorescence can only be detected when dyes are bound to double stranded nucleic acids in the cells.

Sometimes DNA molecules present in scaffold matrices could cause some headache, but that should be only a minor issue.

All the best with your experiments,

Christian

Agreed with Henry E Young, well-explained. However, the 3D-cell culture here is not really clear, are you talking about pre-seeding on the gelatin surface or pre-mixed prior to forming 3D-scaffold, especially hydrogel form. Using an enzymatic approach thru collagenase might work but the time of incubation is really critical to avoid over-digestion that at last may cause cell deterioration..Best of luck!

You should use a solvent-fits drug to extract the drug from the system, then centrifuge, and finally detect it by UV spect. By the way, more details need to be explained to give you the best solution

Thats depend on the methods of fabrication used in my opinion there is no standard for example the 3d printing and electrospinning each give you different parameter. The final decision is made after performing test on your specimens.

Hi,

dear Archana

check the following papers please:

For anisotropic material you might want to look into the Composites module. Regarding which stress to look at, I think that is more a question you should ask yourself, as comsol can give you any stress you want, but you are the one who is in charge of the measurements and your hypotheses.

Hey Sambit! It depends on the fact, which method is the most convinient one for you (and also available at the lab :D). You can for example directly count the cells (by staining their cell nuclei with PI or DAPI, taking the pictures from at least 10 fields of view via fluorescence microscope and manually or via software like MATLAB count them). The other option is to measure their metabolic activity via metabolic assay (MTT, CCK-8,...).

Dear Enric Casanova, the uniformity ans solubility of PCL have strong influence on adsorption. Please check the following documents. My Regards

I think you have not used the pure non wetting solvent ( some percentage of water will be present means its diluted solvent). due to which some of the polar solvent has entered membrane and showing porosity more than its actual porosity.

previously when I cope with gel it also get aspirated. you can use a tip or sth else to press it

Interesting question. Following the discussion.

In order to understand the mechanisms of degradation of biomaterials and release of growth factors, it is necessary to know the chemistry and microstructure, that is, the characterization of the materials and their interaction with the biological medium. After a bone graft, an inflammatory response occurs. and an initial clot, with red blood cells, platelets, and fibrin. Blood clot cells release interleukins and growth factors, causing migration of lymphocytes, macrophages, osteoclast precursors, and MSC (Mesenchymal Stem Cell) pluripotent stem cells. These molecular signals promote the differentiation of undifferentiated cells into endothelial cells, fibroblasts, chondroblasts, and osteoblasts, which will give rise to new fibrovascular tissue, which will replace the initial clot. All this is regulated by a series of complex interactions between growth factors, hormones and cytokines. In this process, vascular supply, protein synthesis and mineralization are essential.

Osteoclasts have two specializations in the membrane: a brush border, which is where reabsorption takes place, and a clear area, rich in microfilaments, with integrins that anchor the matrix. To do this, the osteoclasts are mobilized to the area to be reabsorbed and, subsequently, they adhere to the mineralized bone surface by the brush border, sealing the edges of the area using the integrins. The osteoclast integrin, particularly avβ3, recognizes the Arg-Gly-Asp (RGD) sequence existing in collagen and other osteoid matrix proteins. At this level the pH is acidic, since they secrete acids (H +) generated by carbonic anhydrase II and proteolytic enzymes such as collagenases, metalloproteases, cathepsin K, glucuronidase, etc., which will cause bone resorption by solubilizing the matrix. organic first and mineral later. In a similar way, osteoclasts could act in the dissolution of the biomaterial. Inside the biomaterial, the nanocrystals form a surface microstructure of the graft with negative electrical charges provided by the phosphate groups that are released during the dissolution of the material by cell-material interactions, such as adhesion, proliferation and cell differentiation. Positively charged morphogenetic proteins could either be attracted to this nanometric surface or bind to phosphate groups released into the medium and begin the osteoinduction process. It could be said that nanocrystals and microstructure in synergy with the chemical attraction of phosphate groups released during the dissolution of the biomaterial are mediators or promoters of osteoinduction, but this is only a hypothesis.

Please go to this article :

https://journals.sagepub.com/doi/full/10.1177/2332858419828691

Dominik Hepperle sir thank you for answering my query. I will definitely try it once the software will get downloaded.

Gluing to the bottom won't help; if there's still air in the pores, cells won't get in. The trick is to get it sinking in ethanol or isopropanol first (may need centrifuging or vacuum-release cycles) to get the air out, then put it in PBS or media to wash out the alcohol. Once wetted, it will stay wet and sunk.

The picture you provide isn't the best to represent the difference between contigs and scaffolds. In short:

- you start assembly from short NGS reads

- overlapping parts of the reads are joined to form longer sequences, called the contigs

- For the contigs what you don't know is that if you have 3 contigs, what is their order? Which one presents the beginning of ie. a chromosome, which one is in the middle and which one at the end? Another thing that you don't know is which strand the contig represents? Contig 1 might be on the + strand, contig 2 on the negative strand

- Here comes scaffolding. Using a combination of both already obtained contigs and paired-end read mappings you can put the contigs in their respective order and orientation. Of course, it's not possible for all of the contigs to fall into the scaffolds, in which situation after this step they are scaffolds only by name. Further about the scaffolds, as in the previous step now we don't know the order of the formed scaffolds with respect to one another.

Thanks for your answers Saeed Samani and Bence Ezsias

Hi. What did you try to check? the toxicity of pluronic to MG63 cells? If yes, why the negative control are treated as well with pluronic? I didn't understand the difference between the scaffolds and the negative control. All the best.

Ibtisam Abbas : Thank you so much

I did have a similar issue with Ca2+ crosslinked beads, where I would have them dissolving away or partially losing integrity when washing them with regular PBS. I suppose that PBS having no content of Ca2+ but high concentration of Na+ made the Ca2+ come back to the solution.

I solved the problem by washing the cells with a Ca2+ containing buffer that is compatible with your cells, or simply adding a calcium salt to your PBS. In my case, i washed the cells with DMEM complete, which has a high content of Ca2+, and it maintained the integrity of the beads.

I hope it is of help, best of luck!

Peiran Zhu THANK YOU VERY MUCH FOR YOUR TIME TO ANSWER ME. I WOULD TRY IT !!!

Hi Dilan,

I would favor powders in case you want to be free to work with different solvents. Besides, you can adjust the solution concentrations that may affect the end mechanical properties of the scaffold. You can as well use some other materials like sodium chloride particles in solid form and mix it with the powder to increase the porosity. In the end, you can wash sodium chloride with a poor solvent like water.

I hope it works for you.

Nihan

Dear Raju, thank you for your answer. I agree with you.

Many many thanks for the link.

Regards,

Kaniz

Aaron Haubner thank you ... Alternate ways to create hypoxia seems interesting.

I would suggest XRD because you will get insights about the crystalline structure of the deposits and confirm that you really have apatite. EDX and FTIR are complementary because they will give you information about the chemical composition/structure but are not enough to confirm that you have crystalline apatite.

I hope this helps!

Thank you all.

Good evening

in our puplication we had used adiogenic differentiation of Wharton jeley MSC

As Ajay mentioned, you have to cross linked them with glutaraldehyde. I normally do it with 2% (w/v) in pbs.

remove the medium, add the glutaraldehyde on the cells and leave them like that for 2 hours.

Remove the glutaraldehyde and wash gently with pbs. Then add alcohol 100% and leave it for one 1hr.

Then remove the alcohol and add HMDS (hexamethyldisilazane) to dry the samples overnight and they are ready and completely dried.

now, before SEM, you have to Souter coat them with 5nm of platinium or gold for conductivity. And now, you can finally observe them under SEM

I consider that the first choice (teachers' mediation and assistance, if I understand well) is the case, regarding the main question of the discussion thread.

Dear Jean Raynell Bello

Cancer cells are immortal. Therefore, their use is unsuitable for assessing biocompatibility. According to the international standard, fibroblast cells are used for this task such as L929, SNL and etc. All biological tests must conduct according to ISO standards 10,993–5:1999 (Biological evaluation of medical devices; Part 5: tests for in vitro cytotoxicity). You can read the following articles:

Flexible magnetic polyurethane/Fe2O3 nanoparticles as organic-inorganic nanocomposites for biomedical applications: properties and cell behavior

Preparation and evaluation of polyurethane/cellulose nanowhisker bimodal foam nanocomposites for osteogenic differentiation of hMSCs

Hasan Issa many thanks!

Thank you very much for your answer. The scaffolds are fixed in 4% PFA in PBS1%

The kidneys are obtained from Wistar rats (600-700g). They are around 2 cm after perfusion decellularization.

Hi Jana,

I think it is always better to lyse the samples/ cells first before freezing them for any assays. I would also try flash freezing the samples to retain their biomolecules intact (like flash freezing in liquid nitrogen). Hope this helps. Good luck!

Hi Abdolrasol, I'm just giving you some direction here, not to solve your problem. Think about what an average is xbar=(sum of x)/(count of x). You can find the average velocity formula in any middle school math text book or work it out using differential calculus.

Regards

Ron

آسف السؤال ليس من اختصاصي

Auntie Wikipedia says:

Practically insoluble in water, freely soluble in ethanol, methanol and some other organic solvents. Slightly soluble in vegetable oils. mg/mL (20 °C)

Hi, you can refer this research paper. Hope this will also help.

Röder, A., García-Gareta, E., Theodoropoulos, C., Ristovski, N., Blackwood, K., & Woodruff, M. (2015). An Assessment of Cell Culture Plate Surface Chemistry for in Vitro Studies of Tissue Engineering Scaffolds. Journal of Functional Biomaterials, 6(4), 1054–1063. doi:10.3390/jfb6041054

PGA is more hydrophilic, while PLA is more hydrophobic

Hello Mohammad.

In the past I have done some electrospinning on aluminium foil. To collect some samples I put thin glass coverslips on top which allowed me the collection of the sample (with the aid of a scalpel).

Also, you can try with some teflon tape on top of the aluminium foil, which should detach easily.

Hope it helps,

Hector

Hi Joshua, if you think that your sample might be too diluted you can increase the incubation time. I heard there is a new dye called PrestoBlue that is faster than the Alamar, but works on the same principle. It might be useful in your case.

I believe there is not difference. The mechanism of action is different between UV and Ethanol. UV causes DNA damage, leading inactivation of microorganisms and Ethanol cause denaturation of proteins and membrane dissolution. On the other hand, bacterial spores and some viruses are resistent to both methods. There is not standart methods with parameters approbated by FDA for scaffolds sterilization. The best method depend of chemical composition, size, type, etc.

These two articles are excellent reviews on sterilization of biomaterials.

Regards.

Diego

Thank you Mr. Shavandi, I am going to match this analysis with BCA protein quantification.

Sounds like great work! You can have the students upgrade the e-portfolio to a professional one just on "Educational Design" but for the job market, linking it to their career goals. In that way, they would have artifacts from other their entire program.

Best regards,

Debra

Assuming you have compressed the sample along z-direction and the sample has contracted by dz, then the Poisson ration of 0.3 means that your sample has squashed (swollen) in x- and y-direction by a factor of 0.3dz (provided that your sample is isotropic). Poisson ration is originally defined based on strain forces and indicates how a substance is capable to transfer an exerted force to the transverse directions.

Different Poisson ratios may be attributed to non-identical samples. However, you may check whether your sample is isotropic. That is, you may have measured the Poisson ratio once along x-direction and another time along y-direction.

Hello Usama.

There should be i) a or more sources of stem cells that can generate cartilage cellular and extracellular components, ii) guiding scaffolds that can make a preferable shape in vitro, and iii) other proliferative or inductive growth factors to get sufficient number of differentiated chondrocytes.

Start with the following reviews to grasp a full understanding of current status and limitations.

Best wishes.

Thank you

Great thanking you , I will definitely try it

I too accept with Amin Shavandi answer, In addition to it check the molecular weight of the precursor. If it is very less, even when you increase the concentration gradient you will be resulted with beads.

Firstly I would like to thank you for your sincere recommendations.

Our scaffolds are made of biopolymers (mainly composed of chitosan, cellulose or alginate). Can we detach this mineralized matrix from scaffold surface with Triton x?

Hi Andrea,

Did you mean you freeze dried the scaffold first and then sonicated, and you repeated this three times?

Instead, you can use fluorescent labelled cells. You can trace the cells both in scaffold and their role in wound healing. By the way, what is/are the biomaterials, you are using for the scaffold preparation?

That eill willgive cytocompatability for MSCs, which may suggest compatability compatibilityfir fororger othercelks cellsas well. I would suggest differentiating the cells toward chondrogenic, osteogenic and adipogenic pathways. This will give more info on suitability of the scaffolfscaffol.

Using TGA...

Hi Amin,

thank you for your comment! Indeed plasma modification could be one of the subsequent treatments for the scaffold. However, very often in the beginning of a study we are trying to answer the question how the cells do behave on untreated scaffolds. And as I mentioned above, it is possible to keep cells on the surface when doing simple staining followed by fluorescence microscopy. Most likely, the washing steps in the SEM protocol are too many for loosely attached cells.

I was wondering whether there is a way (trick) to freeze the situation at the end of the experiment. But now I am beginning to think that SEM is probably not the right method to do this sort of analysis (most likely histology would be more suitable) or maybe we should rather extend the contact time of cells with the material from 48h (what is done now) to something like a week. However I am not sure what to expect from the latter option, since it could also be that the whole scaffold will be covered by loosely attached cells.

Thanks Norman. I'll definitely have a look at that going forward.

Dear all,

Usually, apatite signals appear in the fingerprint area of the spectrum. I think they are overlapped somehow by the signal of other groups. Other techniques are more suited such as enhanced resolution FTIR and raman. Regards

I highly recommend you to read this article (related link is below).

chemical characterization and usage area of it studied very well in this article. I hope it will help you to enlight your way.

Kind Regards,

It all comes down to the clinical application and the site that the biomaterials is intended to be used. You can try various composite combinations of different ingredients to see the compatibility of each individual content.

The question is a bit too vague. Why dont you share your materials and share about the intended application so the audience can give you a more specific answer

Das

Try to avoid water in your reaction medium. Here find two methods for similar system

K2CO3 in HOCH2CH2OH polymer for 60 min at rt; see

---Wang, Xi-Cun et al. Synlett, (11), 1657-1660; 2010

or