Product Description

Advanced BioMatrix offers PhotoHA®, a methacrylated hyaluronic acid (HAMA) for photocrosslinkable hydrogels. These hydrogels provide native-like 3D HA gels with the unique attributes to be prepared at various concentrations and photocrosslinked to provide various gel stiffness. The kit comes with 100 mg of lyophilized methacrylated hyaluronic acid and ruthenium and sodium persulfate photoinitiators (400-450 nm visible light photocrosslinking).

Item Catalog Number Package Size Storage Temperature
Methacrylated Hyaluronic Acid #5212 100 mg -20°C
Ruthenium Photoinitiator #5246 100 mg Room Temperature
Sodium Persulfate Photoinitiator #5247 500 mg Room Temperature

 

Hyaluronic acid is the most abundant glycosaminoglycan in the body being an important component of several tissues throughout the body. While it is abundant in extracellular matrices, hyaluronic acid also contributes to tissue hydrodynamics, movement and proliferation of cells, and participates in a number of cell surface receptor interactions.

For the majority of cell types, it is recommended to add additional ECM proteins to the hyaluronic acid hydrogels. The proteins provide important cell binding sites.

Storage:

The product ships on frozen gel packs. Upon receipt, store the PhotoHA® at -20°C. Store the Ruthenium and Sodium Persulfate at room temperature. The product and components are stable for a minimum of 1 year at receipt in powder form.

Once solubilized, the PhotoHA® can be stored at 2-10°C for 1 month. The photoinitiator can be stored for no more than 2 weeks once solubilized.

Parameter, Testing, and Method Methacrylated Hyaluronic Acid #5212
Sterilization Method Filtration
Sterility - USP modified No growth
Form Lyophilized Powder
Package Size 100 mg
Storage Temperature -20°C
Shelf Life Minimum of 6 months from date of receipt
Shelf Life After Reconstitution 1 Month
Degree of Methacrylation 45-65%
Molecular Weight 100-150 kDa

NMR Analysis

Characteristic

Hydrogel Young's Modulus E (Pa)

Characteristic

 

Swelling Characteristics of PhotoHA®:

50 μL hydrogels fabricated in 4.7 mm diameter molds were imaged before and after incubation in phosphate buffered saline at 25°C for 24 hours. The diameter of hydrogels were quantified using ImageJ software. Statistical comparisons between groups (n=3) were performed via one-way ANOVA with post hoc testing and significance determined at p < 0.05. 

 

Compressive Modulus of PhotoHA®:

Dynamic mechanical analysis (Q800, TA Instruments) was performed on 50 μL hydrogels fabricated in 4.7 mm diameter molds. Hydrogels were secured within a fluid cup via a 0.01 N pre-load and compressed to 30% strain at a rate of 0.5 N min-1. The Young’s modulus of each hydrogel was calculated as the slope of generated stress-strain curves between 10% and 20% strain. Statistical comparison between MeHA concentrations (n=3) was performed via Students t-test with two-tailed criteria and significance determined at p < 0.05. 

 

Reaction Behavior of PhotoHA®:

Rheological time sweeps (AR2000 stress controlled rheometer, TA Instruments; 0.5% strain, 1 Hz, 25°C) of MeHA crosslinking with exposure to UV light (=320-390 nm) and in the presence of 0.05 wt% Irgacure 2959 (I2959). After 1 minute, the macromer solution (i.e. MeHA and I2959) was exposed to UV light, resulting in the plateau of moduli before 5 minutes.

Directions for Use

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3D Hydrogel Preparation:

Note: Employ aseptic practices to maintain the sterility of the product throughout the preparation and handling of the collagen and other solutions.

Recommended concentrations are 5-30 mg/ml
(0.5-3.0%).

Note: The following recommended instructions are for a 1% hyaluronic acid (HA) methacrylate solution. Adjustments to this protocol may be required for various concentrations.

  1. Add 10 ml of 1X phosphate buffer saline (PBS), water or cell culture media to the 100 mg of lyophilized methacrylated HA powder.
  2. Mix on a shaker table or rotator plate until fully solubilized (~30 to 60 minutes) at 2-10°C.Note: Solubilization times may vary depending on the desired concentration and volume of PBS, water or medium added.
  3. Calculate the volume of photoinitiator to add by multiplying the volume of solubilized hyaluronic acid by 0.02. If the resulting number is 200 ul, for example, you will add 200 ul of ruthenium and 200 ul of sodium persulfate.
  4. Solubilize the required amount of ruthenium (per step 3) at a concentration of 37.4 mg/ml in 1X PBS or cell culture media.
  5. Solubilize the required amount of sodium persulfate (per step 3) at a concentration of 119 mg/ml in 1X PBS or cell culture media.
  6. Add the ruthenium to the hyaluronic acid solution and fully mix until solution is homogeneous.
  7. Add the sodium persulfate to the hyaluronic acid/ruthenium solution and mix until solution is homogeneous.
  8. Add your cells to the hyaluronic acid/photoinitiator solution.
  9. Dispense your hyaluronic acid/photoinitiator/cell solution into the desired dish (ie. 6-well plate, 48-well plate).
  10. For photocrosslinking, place solution directly under a 400-450 nm visible light crosslinking source.

Product Q & A

Yes. There are quite a few publications citing various hyaluronidase protocols for digesting PhotoHA hydrogels. Here are a few:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5460858/

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4840832/

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4986518/

The HA we use comes from biotechnological production, where the hyaluronic acid is extracted from the cell walls of the bacteria Streptococcus zooepidemicus.

Product Cell Assay

Human mesenchymal stem cells (20 x 106 / mL) were encapsulated in 50 μL hydrogels (~ 4.7 mm x 2 mm). Hydrogels (1 wt% PhotoHA®) were fabricated with 0.05 wt% Irgacure 2959 and exposure to 2 mW/cm2 light (320-390 nm) for 10 minutes.  After 24 hours, encapsulated cells were stained with calcein AM and ethidium homodimer and subsequently imaged on a Leica SP5 confocal microscope (using FITC/TRITC sequential scans).

Product Applications

PhotoHA®  Methacrylated Hyaluronic acid can be used to form hydrogels for ex-vivo engineering of autologous cartilage tissue[1] or as a mesenchymal stem cell carrier in cartilage repair[2].

Because the stiffness can be widely adjusted by altering concentration or UV-light exposure, methacrylated HA has been used to measure the effects of matrix stiffness on cell phenotype and function[3][4].

Methacrylated HA can be used for 3D bioprinting (extrusion[5], inkjet[5] and photolithographic[6]) to create structures that promote osteogenic differentiation of MSC’s[7].

The high tunability of hyaluronic acid methacrylate allows it to be mixed with, and reinforce other types of hydrogels (such as collagen, or gelatin methacrylate)[8].

References:

  1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5717235/

  2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5627486/

  3. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5541838/

  4. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5447944/

  5. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5615317/

  6. https://www.ncbi.nlm.nih.gov/pubmed/21773726/

  7. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5460858/

  8. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5748291/

 

Product References

References for PhotoHA®:

Khetan, Sudhir, and Owen Corey. "Maintenance of stem cell viability and differentiation potential following cryopreservation within 3-dimensional hyaluronic acid hydrogels." Cryobiology(2019).

Poldervaart, M. T. et al. 3D bioprinting of methacrylated hyaluronic acid (MeHA) hydrogel with intrinsic osteogenicity. Plos One12,(2017).

Product Certificate of Analysis

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Safety and Documentation

Safety Data Sheet

Certificate of Origin

Product Disclaimer

This product is for R&D use only and is not intended for human or other uses. Please consult the Material Safety Data Sheet for information regarding hazards and safe handling practices.