Validation of an endothelial roll preparation for Descemet Membrane Endothelial Keratoplasty by a cornea bank using "no touch" dissection technique.

Descemet Membrane Endothelial Keratoplasty (DMEK) selectively replaces the damaged posterior part of the cornea. However, the DMEK technique relies on a manually-performed dissection that is time-consuming, requires training and presents a potential risk of endothelial graft damages leading to surgery postponement when performed by surgeons in the operative room. To validate precut corneal tissue preparation for DMEK provided by a cornea bank in order to supply a quality and security precut endothelial tissue. The protocol was a technology transfer from the Netherlands Institute for Innovative Ocular Surgery (NIIOS) to Lyon Cornea Bank, after formation in NIIOS to the DMEK "no touch" dissection technique. The technique has been validated in selected conditions (materials, microscope) and after a learning curve, cornea bank technicians prepared endothelial tissue for DMEK. Endothelial cells densities (ECD) were evaluated before and after preparation, after storage and transport to the surgery room. Microbiological and histological controls have been done. Twenty corneas were manually dissected; 18 without tears. Nineteen endothelial grafts formed a double roll. The ECD loss after cutting was 3.3 % (n = 19). After transportation 7 days later, we found an ECD loss of 25 % (n = 12). Three days after cutting and transportation, we found 2.1 % of ECD loss (n = 7). Histology found an endothelial cells monolayer lying on Descemet membrane. The mean thickness was 12 ± 2.2 µm (n = 4). No microbial contamination was found (n = 19). Endothelial roll stability has been validated at 3 days in our cornea bank. Cornea bank technicians trained can deliver to surgeons an ECD controlled, safety and ready to use endothelial tissue, for DMEK by "no touch" technique, allowing time saving, quality and security for surgeons.

A Preliminary Study for an Intraoperative 3D Bioprinting Treatment of Severe Burn Injuries.

Intraoperative three-dimensional fabrication of living tissues could be the next biomedical revolution in patient treatment. APPROACH: We developed a surgery-ready robotic three-dimensional bioprinter and demonstrated that a bioprinting procedure using medical grade hydrogel could be performed using a 6-axis robotic arm in vivo for treating burn injuries. RESULTS: We conducted a pilot swine animal study on a deep third-degree severe burn model. We observed that the use of cell-laden bioink as treatment substantially affects skin regeneration, producing in situ fibroblast growth factor and vascular endothelial growth factor, necessary for tissue regeneration and re-epidermalization of the wound. CONCLUSIONS: We described an animal study of intraoperative three-dimensional bioprinting living tissue. This emerging technology brings the first proof of in vivo skin printing feasibility using a surgery-ready robotic arm-based bioprinter. Our positive outcome in skin regeneration, joined with this procedure's feasibility, allow us to envision the possibility of using this innovative approach in a human clinical trial in the near future.

Validation of an implantable bioink using mechanical extraction of human skin cells: First steps to a 3D bioprinting treatment of deep second degree burn.

Clinical grade cultured epithelial autograft (CEA) are routinely used to treat burns covering more than 60% of the total body surface area. However, although the epidermis may be efficiently repaired by CEA, the dermal layer, which is not spared in deep burns, requires additional treatment strategies. Our aim is to develop an innovative method of skin regeneration based on in situ 3D bioprinting of freshly isolated autologous skin cells. We describe herein bioink formulation and cell preparation steps together with experimental data validating a straightforward enzyme-free protocol of skin cell extraction. This procedure complies with both the specific needs of 3D bioprinting process and the stringent rules of good manufacturing practices. This mechanical extraction protocol, starting from human skin biopsies, allows harvesting a sufficient amount of both viable and growing keratinocytes and fibroblasts. We demonstrated that a dermis may be reconstituted in vitro starting from a medical grade bioink and mechanically extracted skin cells. In these experiments, proliferation of the extracted cells can be observed over the first 21 days period after 3D bioprinting and the analysis of type I collagen exhibited a de novo production of extracellular matrix proteins. Finally, in vivo experiments in a murine model of severe burn provided evidences that a topical application of our medical grade bioink was feasible and well-tolerated. Overall, these results represent a valuable groundwork for the design of future 3D bioprinting tissue engineering strategies aimed at treating, in a single intraoperative step, patients suffering from extended severe burns.