Decellularization is a process that strips all the cells from tissue leaving behind the extracellular matrix and its chemical guides. If suitably repopulated with new cells from a patient, the optimal result is living and fully functional tissue matched for transplantation without the possibility of immune rejection. This approach is a stepping stone on the way to generating new complex tissues from scratch, a way to work around the fact that the research community cannot yet produce artificial scaffolds of sufficient complexity and quality to match the natural extracellular matrix structure present in organs. Most work on decellularization to date has involved internal organs such as hearts, livers, and lungs, but there is no reason why it cannot be applied to a limb, as is the case here:
Researchers have used an experimental approach previously used to build bioartificial organs to engineer rat forelimbs with functioning vascular and muscle tissue. They also provided evidence that the same approach could be applied to the limbs of primates. "The composite nature of our limbs makes building a functional biological replacement particularly challenging. Limbs contain muscles, bone, cartilage, blood vessels, tendons, ligaments and nerves - each of which has to be rebuilt and requires a specific supporting structure called the matrix. We have shown that we can maintain the matrix of all of these tissues in their natural relationships to each other, that we can culture the entire construct over prolonged periods of time, and that we can repopulate the vascular system and musculature."
The same decellularization process used in whole-organ studies - perfusing a detergent solution through the vascular system - was used to strip all cellular materials from forelimbs removed from deceased rats in a way that preserved the primary vasculature and nerve matrix. After thorough removal of cellular debris - a process that took a week - what remained was the cell-free matrix that provides structure to all of a limb's composite tissues. At the same time, populations of muscle and vascular cells were being grown in culture.
The research team then cultured the forelimb matrix in a bioreactor, within which vascular cells were injected into the limb's main artery to regenerate veins and arteries. Muscle progenitors were injected directly into the matrix sheaths that define the position of each muscle. After five days in culture, electrical stimulation was applied to the potential limb graft to further promote muscle formation, and after two weeks, the grafts were removed from the bioreactor. Analysis of the bioartificial limbs confirmed the presence of vascular cells along blood vessel walls and muscle cells aligned into appropriate fibers throughout the muscle matrix.
Functional testing of the isolated limbs showed that electrical stimulation of muscle fibers caused them to contract with a strength 80 percent of what would be seen in newborn animals. The vascular systems of bioengineered forelimbs transplanted into recipient animals quickly filled with blood which continued to circulate, and electrical stimulation of muscles within transplanted grafts flexed the wrists and digital joints of the animals' paws. The research team also successfully decellularized baboon forearms to confirm the feasibility of using this approach on the scale that would be required for human patients.