The use of ultrasonication to aid recellularization of acellular natural tissue scaffolds for use in anterior cruciate ligament reconstruction.

Tissue engineering offers a promising solution to the replacement of anterior cruciate ligament. A decellularized porcine patella tendon scaffold was produced by immersing whole tissues sequentially in hypotonic buffer, 0.1% (w/v) sodium dodecyl sulfate (SDS) in hypotonic buffer, and nuclease solution prior to sterilization with 0.1% (w/v) peracetic acid. Initial studies revealed that primary human tenocytes would attach to, but failed to penetrate into, the decellularized scaffold. A novel use of ultrasonication was therefore developed to allow extrinsic cells to migrate into the acellular scaffold. Various intensities of ultrasonication were tested in order to produce a microscopically more open porous matrix without damaging the overall architecture of the scaffold. Ultrasonication treatment with the intensity of 360 W and a pulse time of 1 s for a total of 1 min was found to be the optimal treatment. This process did not have a significant effect upon the biochemical constituents (collagen, glycosaminoglycans), nor did it denature the collagen. Moreover, the acellular sonicated scaffold retained the essential biomechanical characteristics of the native tissue. Primary human tenocytes penetrated into the center of whole acellular sonicated scaffolds over a 3-week period in static culture. The viability of the cells in the center of the scaffold (depth of circa 2.5 mm) was, however, compromised. To circumvent the problem of nutrient limitation, acellular sonicated scaffolds were split into fascicular scaffolds (500 mum thick). Cells seeded onto the fascicular scaffolds penetrated throughout the scaffold and remained viable after 3 weeks of culture. This study has shown that an acellular biocompatible tendon scaffold can be produced using 0.1% (w/v) SDS and that ultrasonication can provide a novel method to enhance the recellularization of decellularized natural tissues.

The effect of amorphous pyrophosphate on calcium phosphate cement resorption and bone generation.

Pyrophosphate ions are both inhibitors of HA formation and substrates for phosphatase enzymes. Unlike polyphosphates their hydrolysis results simultaneously in the complete loss of mineral formation inhibition and a localised elevation in orthophosphate ion concentration. Despite recent advances in our knowledge of the role of the pyrophosphate ion, very little is known about the effects of pyrophosphate on bone formation and even less is known about its local delivery. In this work we first developed a self setting pyrophosphate based calcium cement system with appropriate handling properties and then compared its in vivo degradation properties with those of a non-pyrophosphate containing control. Contrary to expectation, the presence of the pyrophosphate phase in the cement matrix did not inhibit mineralisation of the healing bone around the implant, but actually appeared to stimulate it. In vitro evidence suggested that enzymatic action accelerated dissolution of the inorganic pyrophosphate ions, causing a simultaneous loss of their mineralisation inhibition and a localised rise in supersaturation with respect to HA. This is thought to be a rare example of a biologically responsive inorganic material and these materials seem to be worthy of further investigation. Bioceramics to date have mainly been limited to orthophosphate, silicate and carbonate salts of calcium, here we report the successful application of a pyrophosphate material as a degradable osteoconductive bone repair cement.

The effect of chitin and chitosan on fibroblast-populated collagen lattice contraction.

The effects of chitin [(1-->4)-2-acetamido-2-deoxy-beta-D-glucan] and its partially deacetylated derivatives, chitosans, on the human dermal fibroblast-mediated contraction of collagen lattices were examined in vitro as a model for the contraction of cutaneous wounds in vivo. Chitosan CL313A, a short-chain-length 89% deacetylated chitosan chloride, inhibited fibroblast-populated collagen lattice (FPCL) contraction at higher initial concentrations (500 and 1,000 microg/ml) in FPCLs fabricated with responsive dermal fibroblasts, while in FPCLs containing non-responsive fibroblasts inhibition of contraction was reduced. The responsive and non-responsive phenotype of human dermal fibroblasts to treatment with chitosan CL313A has been reported previously by us. The inhibition of fibroblast-mediated collagen lattice contraction by chitosan appeared to be strongly correlated with whether the cells were responsive or non-responsive. The effect of chitin-50A on fibroblast-mediated collagen lattice contraction was also examined to investigate whether the level of deacetylation was important for its inhibitory effect on contraction. However, this had no effect on contraction at the concentrations tested, supporting previous work that only chitosan samples with higher levels of deacetylation showed any biological activity. This work indicates that highly deacetylated chitosan inhibits fibroblast-mediated contraction of collagen lattices and may therefore be useful as a therapeutic agent to reduce contraction and therefore scarring in wound healing in vivo.