Keratose hydrogel for tissue regeneration and drug delivery.

Keratin (KRT), a natural fibrous structural protein, can be classified into two categories: "soft" cytosolic KRT that is primarily found in the epithelia tissues (e.g., skin, the inner lining of digestive tract) and "hard" KRT that is mainly found in the protective tissues (e.g., hair, horn). The latter is the predominant form of KRT widely used in biomedical research. The oxidized form of extracted KRT is exclusively denoted as keratose (KOS) while the reduced form of KRT is termed as kerateine (KRTN). KOS can be processed into various forms (e.g., hydrogel, films, fibers, and coatings) for different biomedical applications. KRT/KOS offers numerous advantages over other types of biomaterials, such as bioactivity, biocompatibility, degradability, immune/inflammatory privileges, mechanical resilience, chemical manipulability, and easy accessibility. As a result, KRT/KOS has attracted considerable attention and led to a large number of publications associated with this biomaterial over the past few decades; however, most (if not all) of the published review articles focus on KRT regarding its molecular structure, biochemical/biophysical properties, bioactivity, biocompatibility, drug/cell delivery, and in vivo transplantation, as well as its applications in biotechnical products and medical devices. Current progress that is directly associated with KOS applications in tissue regeneration and drug delivery appears an important topic that merits a commentary. To this end, the present review aims to summarize the current progress of KOS-associated biomedical applications, especially focusing on the in vitro and in vivo effects of KOS hydrogel on cultured cells and tissue regeneration following skin injury, skeletal muscle loss, peripheral nerve injury, and cardiac infarction.

Impacts of femoral artery and vein excision versus femoral artery excision on the hindlimb ischemic model in CD-1 mice.

BACKGROUND AND AIM: Although femoral artery ligation-induced ischemia is commonly used in C57BL/6 or Balb/c mice, direct comparisons between femoral artery/vein (FAV) versus femoral artery (FA) excisions have not been reported. The goal of the present study is to investigate the effects of FAV versus FA excisions on hindlimb models using adult CD-1 mice. METHODS: Two groups (n=10/group) of adult, mixed gender CD-1 mice were used to generate hindlimb ischemic models by excising either the FAV or FA. Laser Doppler Imaging was used to evaluate blood flow before surgery, immediately after surgery (Day 0), and then on Days 14 and 28. Toe necrosis was checked every 14days while skeletal muscle cellular remodeling and vascular networks were analyzed at the end of the experiment using pathohistological, Dil-vessel painting, and immunohistochemical approaches. RESULTS: During the 4-week period, no statistical differences were found between FAV and FA excision-induced ischemia in terms of reduction of limb blood flow, paw size, number of necrotic toes, or skeletal muscle cell sizes. However, significant increases in centrally-located nuclei cells, adipose cells, diameters of Dil-stained arterioles, and CD31+ capillary densities, but decreases in arteriole densities/lengths were observed in ischemic limbs of both FAV and FA groups compared to control limbs. CONCLUSION: We conclude that FAV and FA excision in CD-1 mice generate a comparable degree of hindlimb ischemia, suggesting that, as expected, FAV is no more severe than FA. These findings may provide important information for researchers when selecting ligation methods for their hindlimb models.

Polymer microfiber meshes facilitate cardiac differentiation of c-kit(+) human cardiac stem cells.

Electrospun microfiber meshes have been shown to support the proliferation and differentiation of many types of stem cells, but the phenotypic fate of c-kit(+) human cardiac stem cells (hCSCs) have not been explored. To this end, we utilized thin (~5µm) elastomeric meshes consisting of aligned 1.7µm diameter poly (ester-urethane urea) microfibers as substrates to examine their effect on hCSC viability, morphology, proliferation, and differentiation relative to cells cultured on tissue culture polystyrene (TCPS). The results showed that cells on microfiber meshes displayed an elongated morphology aligned in the direction of fiber orientation, lower proliferation rates, but increased expressions of genes and proteins majorly associated with cardiomyocyte phenotype. The early (NK2 homeobox 5, Nkx2.5) and late (cardiac troponin I, cTnI) cardiomyocyte genes were significantly increased on meshes (Nkx=2.5 56.2±13.0, cTnl=2.9±0.56,) over TCPS (Nkx2.5=4.2±0.9, cTnl=1.6±0.5, n=9, p<0.05 for both groups) after differentiation. In contrast, expressions of smooth muscle markers, Gata6 and myosin heavy chain (SM-MHC), were decreased on meshes. Immunocytochemical analysis with cardiac antibody exhibited the similar pattern of above cardiac differentiation. We conclude that aligned microfiber meshes are suitable for guiding cardiac differentiation of hCSCs and may facilitate stem cell-based therapies for treatment of cardiac diseases.

Superior angiogenesis facilitates digit regrowth in MRL/MpJ mice compared to C57BL/6 mice.

Previous studies indicated that the fast-healer strain of MRL/MpJ-Fas(lpr)/J (MRL) mice demonstrated superior regenerative capabilities for digit wound healing and/or regeneration compared with the non-healer strain of C57BL/6 (C57) mice. These reports, however, mainly focused on morphological observations and analysis of gene expression with little attention on the role of angiogenesis in the amputated digits. By taking advantage of Laser Doppler Imaging and histological analysis, we examined the potential role(s) of angiogenesis in facilitating tissue regrowth/regeneration by comparing two strains of mice (MRL versus C57). The three middle digits on the mouse's right foot (RF) were amputated at the middle level of phalanx 2 (P2) on postnatal day 2 (Day 0), while the left foot (LF) remained intact and served as a control. Laser Doppler images and digital photographs were taken of both feet before, immediately after surgery, and on Day 7, 14, 21, and 28 to evaluate blood flow and overall length of digit regrowth. All measurements from the amputated digits of the RF were divided by those of the control LF to obtain normalized ratios for statistical comparisons between groups. It was found that MRL mice demonstrated an approximately 220% increase in regrowth ratios over that of C57 mice from Day 21-28 (p < 0.01, n = 13), while blood-flow increased by about 25% on Day 21 (p < 0.01, n = 13) compared to that in C57 mice. Histological analysis of both control and amputated limbs indicated an approximately 70% increase in the number of vessels (both arterial and venous) in MRL mice over that of the C57 mice (p < 0.05, n = 3). We conclude that higher blood flow and angiogenesis may play an important role in facilitating the fast regrowth ratios of amputated digits in MRL mice compared to C57 mice.

Degradation of the α-Carboxyl Terminus 11 Peptide: In Vivo and Ex Vivo Impacts of Time, Temperature, Inhibitors, and Gender in Rat.

In previous research, a synthetic α-carboxyl terminus 1 (αCT1) peptide derived from connexin 43 (Cx43) and its variant (αCT11) showed beneficial effects in an ex vivo ischemia-reperfusion (I/R) heart injury model in mouse. In an in vivo mouse model of cryo-induced ventricular injury, αCT1 released from adhesive cardiac patches reduced Cx43 remodeling and arrhythmias, as well as maintained cardiac conduction. Whether intravenous injection of αCT1 or αCT11 produces similar outcomes has not been investigated. Given the possibility of peptide degradation in plasma, this study utilized in vivo I/R cardiac injury and ex vivo blood plasma models to examine factors that may limit the therapeutic potential of peptide therapeutics in vivo. Following tail vein administration of αCT11 (100 µM) in blood, no effect on I/R infarct size was observed in adult rat hearts on day 1 (D1) and day 28 (D28) after injury (p > 0.05). There was also no difference in the echocardiographic ejection fraction (EF%) between the control and the αCT11 groups (p > 0.05). Surprisingly, αCT11 in blood plasma collected from these rats was undetectable within ∼10 min after tail vein injection. To investigate factors that may modulate αCT11 degradation in blood, αCT11 was directly added to blood plasma isolated from normal rats without I/R and peptide levels were measured under different experimental conditions. Consistent with in vivo observations, significant αCT11 degradation occurred in plasma within 10 min at 22 and 37 °C and was nearly undetectable by 30 min. These responses were reduced by the addition of protease/phosphatase (PTase/PPTase) inhibitors to the isolated plasma. Interestingly, no significant differences in αCT11 degradation in plasma were noted between male and female rats. We conclude that fast degradation of αCT11 is likely the reason that no beneficial effects were observed in the in vivo I/R model and inhibition or shielding from PTase/PPTase activity may be a strategy that will assist with the viability of peptide therapeutics.

Keratose Hydrogel Drives Differentiation of Cardiac Vascular Smooth Muscle Progenitor Cells: Implications in Ischemic Treatment.

Peripheral artery disease (PAD) is a common vascular disorder in the extremity of limbs with limited clinical treatments. Stem cells hold great promise for the treatment of PAD, but their therapeutic efficiency is limited due to multiple factors, such as poor engraftment and non-optimal selection of cell type. To date, stem cells from a variety of tissue sources have been tested, but little information is available regarding vascular smooth muscle cells (VSMCs) for PAD therapy. The present study examines the effects of keratose (KOS) hydrogels on c-kit+/CD31- cardiac vascular smooth muscle progenitor cell (cVSMPC) differentiation and the therapeutic potential of the resultant VSMCs in a mouse hindlimb ischemic model of PAD. The results demonstrated that KOS but not collagen hydrogel was able to drive the majority of cVSMPCs into functional VSMCs in a defined Knockout serum replacement (SR) medium in the absence of differentiation inducers. This effect could be inhibited by TGF-ß1 antagonists. Further, KOS hydrogel increased expression of TGF-ß1-associated proteins and modulated the level of free TGF-ß1 during differentiation. Finally, transplantation of KOS-driven VSMCs significantly increased blood flow and vascular densities of ischemic hindlimbs. These findings indicate that TGF-ß1 signaling is involved in KOS hydrogel-preferred VSMC differentiation and that enhanced blood flow are likely resulted from angiogenesis and/or arteriogenesis induced by transplanted VSMCs.

Peptide Amphiphile Supramolecular Nanofibers Designed to Target Abdominal Aortic Aneurysms.

An abdominal aortic aneurysm (AAA) is a localized dilation of the aorta located in the abdomen that poses a severe risk of death when ruptured. The cause of AAA is not fully understood, but degradation of medial elastin due to elastolytic matrix metalloproteinases is a key step leading to aortic dilation. Current therapeutic interventions are limited to surgical repair to prevent catastrophic rupture. Here, we report the development of injectable supramolecular nanofibers using peptide amphiphile molecules designed to localize to AAA by targeting fragmented elastin, matrix metalloproteinase 2 (MMP-2), and membrane type 1 matrix metalloproteinase. We designed four targeting peptide sequences from X-ray crystallographic data and incorporated them into PA molecules via solid phase peptide synthesis. After coassembling targeted and diluent PAs at different molar ratios, we assessed their ability to form nanofibers using transmission electron microscopy and to localize to AAA in male and female Sprague-Dawley rats using light sheet fluorescence microscopy. We found that three formulations of the PA nanofibers were able to localize to AAA tissue, but the MMP-2 targeting PA substantially outperformed the other nanofibers. Additionally, we demonstrated that the MMP-2 targeting PA nanofibers had an optimal dose of 5 mg (∼12 mg/kg). Our results show that there was not a significant difference in targeting between male and female Sprague-Dawley rats. Given the ability of the MMP-2 targeting PA nanofiber to localize to AAA tissue, future studies will investigate potential diagnostic and targeted drug delivery applications for AAA.

Development of Poly(1,8-octanediol-co-citrate-co-ascorbate) Elastomers with Enhanced Ascorbate Performance for Use as a Graft Coating to Prevent Neointimal Hyperplasia.

Small-diameter expanded polytetrafluoroethylene (ePTFE) graft surfaces have poor long-term patency due to limited endothelial cell (EC) coverage and anastomotic intimal hyperplasia. Multifunctional elastomers that coat the ePTFE graft surface to promote EC adhesion while simultaneously inhibiting intimal hyperplasia are highly desirable. Poly(diol-co-citrate) (PDC), a thermoset elastomer, is biodegradable, biocompatible, and mimics vascular mechanical properties. Engineering antioxidant components into PDC polymeric structures improves biocompatibility by attenuating oxidative stress yet is limited by bioavailability. Herein, we develop a new ascorbate protection and deprotection strategy (APDS) for loading bioactive ascorbic acid into the structure of PDC elastomers to improve poly(1,8-octanediol-co-citrate-co-ascorbate) (POCA) prepolymer ascorbate activity. Elastomers cured from APDS POCA prepolymers provide twice the active ascorbate sites on the elastomer surface (35.19 ± 1.64 ng mg-1 cm-2) versus unprotected POCA (Un.POCA, 18.31 ± 0.97 ng mg-1 cm-2). APDS POCA elastomers displayed suitable mechanical properties for vascular graft coating [Young's modulus (2.15-2.61 MPa), elongation (189.5-214.6%) and ultimate tensile strength (2.73-3.61 MPa)], and superior surface antioxidant performance through 1,1-diphenyl-2-picrylhydrazyl free radical scavenging and lipid peroxidation inhibition as compared to poly(1,8-octanediol-co-citrate) (POC) and Un.POCA. Hydrolytic degradation of APDS POCA occurred within 12 weeks under physiological conditions with a mass loss of 25.8 ± 3.4% and the degradation product retaining ascorbate activity. APDS POCA elastomer surfaces supported human aortic endothelial cell proliferation while inhibiting human aortic smooth muscle cell proliferation in vitro. APDS POCA elastomer surfaces displayed superior decomposition of S-nitrosothiols compared to POC and Un.POCA. Taken together, these findings indicate the potential of APDS POCA elastomers to serve as bioactive, therapeutic coatings that enhance the long-term patency of small diameter ePTFE grafts.

Atheroma Niche-Responsive Nanocarriers for Immunotherapeutic Delivery.

Nanomedicine is a promising, noninvasive approach to reduce atherosclerotic plaque burden. However, drug delivery is limited without the ability of nanocarriers to sense and respond to the diseased microenvironment. In this study, nanomaterials are developed from peptide amphiphiles (PAs) that respond to the increased levels of matrix metalloproteinases 2 and 9 (MMP2/9) or reactive oxygen species (ROS) found within the atherosclerotic niche. A pro-resolving therapeutic, Ac2-26, derived from annexin-A1 protein, is tethered to PAs using peptide linkages that cleave in response to MMP2/9 or ROS. By adjusting the molar ratios and processing conditions, the Ac2-26 PA can be co-assembled with a PA containing an apolipoprotein A1-mimetic peptide to create a targeted, therapeutic nanofiber (ApoA1-Ac226 PA). The ApoA1-Ac2-26 PAs demonstrate release of Ac2-26 within 24 h after treatment with MMP2 or ROS. The niche-responsive ApoA1-Ac2-26 PAs are cytocompatible and reduce macrophage activation from interferon gamma and lipopolysaccharide treatment, evidenced by decreased nitric oxide production. Interestingly, the linkage chemistry of ApoA1-Ac2-26 PAs significantly affects macrophage uptake and retention. Taken together, these findings demonstrate the potential of PAs to serve as an atheroma niche-responsive nanocarrier system to modulate the inflammatory microenvironment, with implications for atherosclerosis treatment.

Keratose Hydrogels Promote Vascular Smooth Muscle Differentiation from C-kit-Positive Human Cardiac Stem Cells.

Stem cell-based therapies have demonstrated great potential for the treatment of cardiac diseases, for example, myocardial infarction; however, low cell viability, low retention/engraftment, and uncontrollable in vivo differentiation after transplantation are still major limitations, which lead to low therapeutic efficiency. Biomaterials provide a promising solution to overcome these issues due to their biocompatibility, biodegradability, low/nonimmunogenicity, and low/noncytotoxicity. The present study aimed to investigate the impacts of keratose (KOS) hydrogel biomaterial on cellular viability, proliferation, and differentiation of c-kit+ human cardiac stem cells (hCSCs). Briefly, hCSCs were cultured on both KOS hydrogel-coated dishes and regular tissue culture dishes (Blank control). Cell viability, stemness, proliferation, cellular morphology, and cardiac lineage differentiation were compared between KOS hydrogel and the Blank control at different time points. We found that KOS hydrogel is effective in maintaining hCSCs without any observable toxic effects, although cell size and proliferation rate appeared smaller on the KOS hydrogel compared to the Blank control. To our surprise, KOS hydrogel significantly promoted vascular smooth muscle cell (VSMC) differentiation (∼72%), while on the Blank control dishes, most of the hCSCs (∼78%) became cardiomyocytes. Furthermore, we also observed "endothelial cell tube-like" microstructures formed by differentiated VSMCs only on KOS hydrogel, suggesting a potential capability of the hCSC-derived VSMCs for in vitro angiogenesis. To the best of our knowledge, this is the first report to discover the preferred differentiation of hCSCs toward VSMCs on KOS hydrogel. The underlying mechanism remains unknown. This innovative methodology may offer a new approach to generate a robust number of VSMCs simply by culturing hCSCs on KOS hydrogel, and the resulting VSMCs may be used in animal studies and clinical trials in combination with an injectable KOS hydrogel to treat cardiovascular diseases.

Tattooing Various Combinations of Ears, Tail, and Toes to Identify Mice Reliably and Permanently.

Uniquely identifying research animals is a widespread and essential procedure. Potential disadvantages of commonly used identification methods such as toe clipping, ear punching, and ear tagging include tissue loss and adverse effects in physiologic homeostasis and animal behavior. In addition, the labels produced by using these methods can become unreadable, potentially leading to misidentification. In this study, we proposed a combined approach involving ear, tail, and toe tattooing that can be used to permanently identify mice regardless of their age. Four groups (neonatal and adult C57BL/6J [black] and CD1 [white] mice) were used. Single- or 2-color tattooing (ear, tail, or toe or combinations thereof) was performed to identify a defined or unlimited number of mice, respectively. Tail tattooing using both green and red pastes was suitable for identifying white-haired neonatal mice as early as postnatal day 1, whereas toe tattooing with green paste was an effective alternative approach for labeling black-haired mouse pups. In comparison, single-color (green) or 2-color (green and red) ear tattooing identified both white and black adult mice older than 3 wk. Ear tattooing can be adapted to labeling an unlimited number of adult mice by adding the cage number. We conclude that tattooing various combinations of the ears, tail, and toes provides an easy and permanent approach for identifying mice of all ages with minimal disturbance to the animals.

Rho-Associated Kinase Inhibitor (Y-27632) Attenuates Doxorubicin-Induced Apoptosis of Human Cardiac Stem Cells.

BACKGROUND: Recent clinical trials using c-kit+ human cardiac stem cells (CSCs) demonstrated promising results in increasing cardiac function and improving quality of life. However, CSC efficiency is low, likely due to limited cell survival and engraftment after transplantation. The Rho-associated protein kinase (ROCK) inhibitor, Y-27632, significantly increased cell survival rate, adhesion, and migration in numerous types of cells, including stem cells, suggesting a common feature of the ROCK-mediated apoptotic pathway that may also exist in human CSCs. In this study, we examine the hypothesis that pretreatment of human CSCs with Y-27632 protects cells from Doxorubicin (Dox) induced apoptosis. METHODS AND RESULTS: c-kit+ CSCs were cultured in CSC medium for 3-5 days followed by 48 hr treatment with 0 to 10 µM Y-27632 alone, 0 to 1.0 µM Dox alone, or Y-27632 followed by Dox (48 hrs). Cell viability, toxicity, proliferation, morphology, migration, Caspase-3 activity, expression levels of apoptotic-related key proteins and c-kit+ were examined. Results showed that 48 hr treatment with Y-27632 alone did not result in great changes in c-kit+ expression, proliferation, Caspase-3 activity, or apoptosis; however cell viability was significantly increased and cell migration was promoted. These effects likely involve the ROCK/Actin pathways. In contrast, 48 hr treatment with Dox alone dramatically increased Caspase-3 activity, resulting in cell death. Although Y-27632 alone did not affect the expression levels of apoptotic-related key factors (p-Akt, Akt, Bcl-2, Bcl-xl, Bax, cleaved Caspase-3, and Caspase-3) under basal conditions, it significantly inhibited the Dox-induced increase in cleaved Caspase-3 and reduced cell death under Dox treatment. CONCLUSIONS: We conclude that preconditioning human CSCs with Y-27632 significantly reduces Dox-induced cell death and possibly involves the cleaved Caspase-3 and ROCK/Actin pathways. The beneficial effects of Y-27632 may be applied to stem cell-based therapy to increase cell survival rates after transplantation or to act as a cardiac protective agent for Dox-treated cancer patients.