Biodegradation

In vivo degradation of Extracel™

Extracel is naturally degraded by endogenous hyaluronidases (enzymes that breakdown the large hyaluronan polymer into small fragments – see below for details) and collagenases produced by cells. Resorption time in vivo depends on the solids concentration of the implanted Extracel, the form in which it is implanted, and whether cells are encapsulated in the material¹. Hydrogels implanted without any cells persist much longer in vivo than those used to encapsulate cells¹. With cells, standard Extracel formulation hydrogels persist in vivo for up to 4 weeks, while lyophilized sponges of the same material may last as long as 8 weeks in the body^1,2,3,4. In general, hydrogels degrade faster than lyophilized sponges, and lowering the solids’ concentrations decreases the degradation time for either construct. This versatility allows researchers to deliberately vary the in vivo degradation times based on their experimental requirements.

The Extracel hydrogels and sponges are readily visible when the tissue resulting from the implant is removed, fixed and stained⁵. It is also possible to track the persistence of the hydrogels by adding a fluorescent dye prior to implantation (see protocol “Fluorescent labeling”).

In vitro degradation of Extracel

Extracel is composed of modified Glycosil™ (thiol-modified hyaluronan, HA) and Gelin-S™ (thiol-modified gelatin, denatured collagen) chemically crosslinked with Extralink™ (polyethylene glycol diacrylate). Hyaluronidases and collagenases can be used to degrade the Extracel hydrogels in vitro and are commercially available. For details on these experiments, see reference 1. Without enzymatic digestion, the Extracel hydrogels persist in PBS for well over 50 days¹.

Hyaluronidases

There are three classifications of hyaluronidases that digest HA⁶:

mammalian (endo-β-N-acetyl-D-hexosaminidases that make tetra- and hexasaccharides)
leeches/parasite (endo-β-glucuronidases)
bacterial (act through β-elimination to make di-, tetra- or hexasaccharides; note: this enzyme introduces a double bond in the uronic acid at the non-reducing end which is detectable at 232 nm).

Hyaluronidases function best at acidic pH, which is expected given their role in lyosomal degradation. Enzymatic digestions are typically done in sodium acetate buffer (pH 4.8-6.0) at 37 °C. By varying the time of digestion, the resulting pool of HA oligosaccharides changes – the longer the digestion, the shorter the chain lengths⁶.

There are 5 homologous mammalian types of hyaluronidases encoded in the human genome: Hyal-1 to 4 and PH-20 (sperm adhesion molecule 1, or SPAM-1)⁷.

Hyal-1 is expressed in most tissues as well as being detected in plasma and urine. Hyal-1 is not membrane bound; however, it requires CD44 for hyaluronidase activity in vivo⁷.
Hyal-2 is also expressed in most tissues; however, it is absent from the brain. It breaks down extracellular HA. It possesses a glycosylphosphatidylinositol (GPI) signal sequence, which anchors it to the membrane. Hyal-2 also requires CD44 to be able to degrade HA in vivo⁷. It is thought to be an extracellular enzyme that is key for tissue remodeling and cellular migration⁸.
Hyal-3 is expressed in brain and several other tissues, but its function has not been determined. It is GPI anchored⁷.
Hyal-4 is specific for chondroitin sulfate substrates and is GPI anchored⁷.
PH-20 is expressed in sperm and is active during fertilization where it degrades the HA-enriched cumulus of the egg^7,8.

Systemic removal of hyaluronan

HA is removed from an organism by two routes, both of which require hyaluronidases^7,8:

1. Internalization and degradation by cells and destruction in the lyosome⁸:

The hyaluronidase, Hyal-1, is responsible for the catabolism of intracellular HA and functions primarily in the lyosome^7,8. This is the enzyme that creates angiogenic HA fragments since it cleaves HA chains of all sizes down to tetrasaccharides⁷. Degradation of the tetrasaccharides created by Hyal-1 is completed by β-glucuronidases and β-N-acetyl-glucosaminidases in the lyosome. The final degradation products are GlcNAc (which can be recycled) and GlcA (which is catabolized in the pentose pathway)⁹.

2. Release from the ECM and drainage into the vasculature, followed by removal by the lymph nodes, liver and kidney⁸. The hyaluronidase, Hyal-2, is responsible for breaking down extracellular HA^7,8. After being cleaved, HA is transported through the lymphatic system. Once in the blood stream, the liver removes about 80% and the kidney another 10%⁸.

References