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Aims: Normal lungs do not express alpha-Klotho (Klotho) protein but derive cytoprotection from circulating soluble Klotho. It is unclear whether chronic supranormal Klotho levels confer additional benefit. To address this, we tested the age-related effects of Klotho overexpression on acute lung injury (ALI) and recovery. Methods: Transgenic Klotho-overexpressing (Tg-Kl) and wild-type (WT) mice (2 and 6 months old) were exposed to hyperoxia (95% O2; 72 h) then returned to normoxia (21% O2; 24 h) (Hx-R). Control mice were kept in normoxia. Renal and serum Klotho, lung histology, and bronchoalveolar lavage fluid oxidative damage markers were assessed. Effects of hyperoxia were tested in human embryonic kidney cells stably expressing Klotho. A549 lung epithelial cells transfected with Klotho cDNA or vector were exposed to cigarette smoke; lactate dehydrogenase and double-strand DNA breaks were measured. Results: Serum Klotho decreased with age. Hyperoxia suppressed renal Klotho at both ages and serum Klotho at 2-months of age. Tg-Kl mice at both ages and 2-months-old WT mice survived Hx-R; 6-months-old Tg-Kl mice showed lower lung damage than age-matched WT mice. Hyperoxia directly inhibited Klotho expression and release in vitro; Klotho transfection attenuated cigarette smoke-induced cytotoxicity and DNA double-strand breaks in lung epithelial cells. Conclusions: Young animals with chronic high baseline Klotho expression are more resistant to ALI. Chronic constitutive Klotho overexpression in older Tg-Kl animals attenuates hyperoxia-induced lung damage and improves survival and short-term recovery despite an acute reduction in serum Klotho level during injury. We conclude that chronic enhancement of Klotho expression increases resilience to ALI.

Circulating alpha-Klotho produced mainly by the kidney is an essential pleiotropic protein for tissue maintenance, anti-senescence, antioxidation and protection from injury. Normal murine and human lungs do not express Klotho, but critically depend on circulating Klotho for antioxidation and protection from acute lung Injury (ALI). Homozygous Klotho deficient (kl/kl) mice exhibit heightened oxidative stress damage in the lung along with premature widespread organ degeneration and early death. Acquired Klotho deficiency caused by acute kidney injury exacerbates the development of secondary ALI while exogenous Klotho repletion ameliorates ALI, suggesting that increasing circulating Klotho level could protect lungs from ALI. We bred transgenic mice (Tg-Kl, 129sv) that overexpress transmembrane Klotho protein under the control of a ubiquitous human elongation factor-1-alpha promoter (EFmKL46) and exhibit chronically elevated serum Klotho level compared to wild type (WT) mice (40.9±7.7 vs. 18.0±7.1 pmol/L, respectively, mean±SD). We tested the hypothesis that Tg-Kl mice are more resistant to hyperoxia-induced ALI compared to WT mice. METHODS: Adult mice (6 mo. Old) were exposed to hyperoxia (Hx, 95% O 2 ) in an environment chamber for 72 h, then removed to normoxia (Nx, 21% O 2 ) for 24 h.Corresponding Tg-Kl and WT mice were kept in Nx as controls (n=10/group, equal male/female). Bronchoalveolar Lavage fluid (BALF) was assayed for total protein, total antioxidant capacity (copper-reducing equivalents, CRE), and oxidative stress damage markers (8-isoprostane, 8-hydroxy-2'-deoxyguanosine , and protein carbonyl). One lung was fixed (25 cmH 2 O airway pressure) for histology. RESULTS: Four Hx WT mice died; there were no deaths among Hx Tg-Kl mice. In Hx WT mice, total protein, CRE, and all oxidative damage biomarkers were markedly elevated compared to that in Nx; in Tg-Kl mice the corresponding Hx-induced increases were uniformly attenuated (Table ). Distal lung morphology was better preserved in Hx Tg-Kl mice with less alveolar septal thickening, edema and exudation compared to Hx WT mice. CONCLUSION: Klotho overexpression protects lungs from acute oxidant injury, suggesting that interventions to increase serum Klotho level may have translational value in the prevention and/or alleviation of ALI.

Stem cells have become a revived biotechnology that is beginning to expand the field of regenerative medicine. Although stem cells are capable of regenerating tissues, current research trends tend to side on developing fully functional organs and other clinical uses including in situ stem cell repair through three-dimensional printing methods. Through several tests and techniques, it can be shown that most stem cell printing methods are possible and that most tests come out with high cell viability. Furthermore, the importance of bioprinting is to benefit the field of regenerative medicine, which looks into artificial organ transplants for the thousands of patients without donors. Although the field is not brand new, understanding the integration and use of additive manufacturing with biomaterials is essential in developing fully functional organs. There is a heavy emphasis on the biomaterials themselves since they have a crucial role in creating an organ that is mechanically robust and adaptable in vivo. Covered in this review article are many featured tests, which also touch on the importance of including a biomaterial that is capable of maintaining a viable microenvironment. These include biomaterials such as hydrogels, biopolymers, and synthetic extra cellular matrices (ECM) built for stem cells to proliferate, differentiate, and give freedom to cell communication after printing.

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