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Creating Bioartificial Limb Replacements - Give us a hand!

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Review of "Engineered composite tissue as a bioartificial limb graft" from Biomaterials by Stuart P. Atkinson

Recent studies have described how decellularized organs can serve as extracellular matrix (ECM) scaffolds for repopulation with various stem and somatic cells as a strategy to create replacements for simple organs and tissues [1, 2]. The application of this strategy to complex organs and appendages has not been as successful, due to the lack of suitable scaffolds and the difficulty in repopulating these with different cell types. In an exciting step forward, researchers from the laboratory of Harald C. Ott (Harvard) have recently described the successful creation of rat forearm grafts. Their strategy involved repopulating acellular rat limb scaffolds with cells of the muscle and vasculature followed by maturation in a perfusion bioreactor under electrical stimulation in vitro. This yielded a bio-composite graft which became perfused with the recipients circulation after transplantation, and may lay the groundwork towards creating complex human limb grafts [3].

To remove cellular material from the rat limb to create the scaffold, after removing the skin and severing connective tissue to inhibit fluid buildup and scaffold deterioration the researchers used a slow low-pressure detergent based perfusion protocol. This mediated the highly efficient removal of cellular tissue components but it also preserved the composite tissue architecture and extracellular matrix structure of muscles, tendons, bones, ligaments, nerves and blood vessels. The optimized length of the process inhibited the removal of ECM components, such as sulfated glycosaminoglycans, which can affect the biomechanical properties and biochemical composition of engineered grafts. Analyses of the acellular scaffold found no weakening of the skeletal system (mechanical, mineral or geometric bone characteristics), the continued presence of perfusable vascular channels throughout the entire graft, and the functional preservation of the entire skeletomuscular system.

To recellularize the graft, the group first infused vascular endothelial cells into the brachial artery of the scaffold to generate a homogenously distributed vasculatory system, followed by immediate culture in a perfusion bioreactor. The day after, the building blocks of contractile muscle were introduced by injecting myoblasts, fibroblasts and endothelial cells into the muscle compartment using small diameter cannulas under a surgical microscope. Initial cell grafting utilized a low glucoses DMEM-based medium which was then changed to low-serum differentiation medium which allowed for myotube fusion and the electric field-mediated formation of functional muscle. The researchers than added full thickness skin grafts on day 10, and cultured the construct until day 21 at which time histological analysis found that seeded cells had engrafted in their appropriate compartments, displayed the expected behavior, and expressed typical markers. Furthermore, the grafts demonstrated good functionality, with engineered muscle fibers generating a force at around 80% of that observed for neonatal muscle. Finally, the group was able to transplant the engineered limb and demonstrated adequate blood flow between the host and the transplant and the preservation of mobility and functional potential following electrical stimulation of the recipient's forearm muscles.

This exciting new study will pave the way for further improvements and refinements which may soon provide complex transplantable organs and limbs. The nervous system of grafts still requires attention and, indeed, the authors suggest that as recipient nerves have to regrow into the donor nerve sheaths, enhanced functional maturation may require the transplantation of more immature limb replacements, allowing them to mature whilst forming an appropriate connection with the host. The authors also demonstrated that their decellularization protocol could generate an ECM scaffold from a primate forearm, suggesting that the process can potentially be scaled to a clinically relevant size, and may be applicable to human limb replacement therapies.

References

  1. Ott HC, Clippinger B, Conrad C, et al. Regeneration and orthotopic transplantation of a bioartificial lung. Nature Medicine 2010;16:927-933.
  2. Ott HC, Matthiesen TS, Goh SK, et al. Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart. Nature Medicine 2008;14:213-221.
  3. Jank BJ, Xiong L, Moser PT, et al. Engineered composite tissue as a bioartificial limb graft. Biomaterials 2015;61:246-256.