In Vitro and In Vivo Studies of Biodegradability and Biocompatibility of Poly(εCL)-b-Poly(EtOEP)-Based Films

The control of surface bioadhesive properties of the subcutaneous implants is essential for the development of biosensors and controlled drug release devices. Poly(alkyl ethylene phosphate)-based (co)polymers are structurally versatile, biocompatible and biodegradable, and may be regarded as an alternative to poly(ethylene glycol) (PEG) copolymers in the creation of antiadhesive materials. The present work reports the synthesis of block copolymers of ε-caprolactone (εCL) and 2-ethoxy-1,3,2-dioxaphospholane-2-oxide (ethyl ethylene phosphate, EtOEP) with different content of EtOEP fragments, preparation of polymer films, and the results of the study of the impact of EtOEP/εCL ratio on the hydrophilicity (contact angle of wetting), hydrolytic stability, cytotoxicity, protein and cell adhesion, and cell proliferation using umbilical cord multipotent stem cells. It was found that the increase of EtOEP/εCL ratio results in increase of hydrophilicity of the polymer films with lowering of the protein and cell adhesion. MTT cytotoxicity test showed no significant deviations in toxicity of poly(εCL) and poly(εCL)-b-poly(EtOEP)-based films. The influence of the length of poly(EtOEP)chain in block-copolymers on fibrotic reactions was analyzed using subcutaneous implantation experiments (Wistar line rats), the increase of the width of the fibrous capsule correlated with higher EtOEP/εCL ratio. However, the copolymer-based film with highest content of polyphosphate had been subjected to faster degradation with a formation of developed contact surface of poly(εCL). The rate of the degradation of polyphosphate in vivo was significantly higher than the rate of the degradation of polyphosphate in vitro, which only confirms an objective value of in vivo experiments in the development of polymer materials for biomedical applications

Umbilical cord-derived mesenchymal stromal/stem cells enhance recovery of surgically induced skeletal muscle ischemia in a rat model

This study delves into possible mechanisms underlying the stimulating influence of UC-MSCs transplantation on functional and structural recovery of ischemic skeletal muscles. Limb ischemia was created in Sprague-Dawley rats by excision of femoral and popliteal arteries. Allogeneic rat PKH26-labeled UCMSCs were administered by direct intramuscular injection. Animals of experimental group responded to the transplantation by improvement in their locomotor function as assessed by the rotarod performance test on day 9 and 29 after transplantation. Histomorphometric analysis showed that relative area of the lesions in the experimental group was significantly smaller than in the control group at all time points during the observation. Calculated densities of microcirculation vessels within the lesions were significantly higher in the experimental group than in the control group on day 10 after transplantation. Only a part of the transplanted allogeneic UC-MSCs survived within the ischemic muscle tissue, and a considerable portion of these surviving cells were found alongside the VEGFproducing preserved muscle fibers. The РКН26 label was not found within the walls of capillaries or larger blood vessels. The administration of allogeneic UCMSCs significantly increased the proportion of M2 macrophages, exhibiting proangiogenic and antiinflammatory properties, for at least 10 days following the transplantatio

Molecular Survey of Cell Source Usage during Subtotal Hepatectomy-Induced Liver Regeneration in Rats

<div><p>Proliferation of hepatocytes is known to be the main process in the hepatectomy-induced liver regrowth; however, in cases of extensive loss it may be insufficient for complete recovery unless supported by some additional sources e.g. mobilization of undifferentiated progenitors. The study was conducted on rat model of 80% subtotal hepatectomy; the objective was to evaluate contributions of hepatocytes and resident progenitor cells to the hepatic tissue recovery via monitoring specific mRNA and/or protein expression levels for a panel of genes implicated in growth, cell differentiation, angiogenesis, and inflammation. Some of the genes showed distinctive temporal expression patterns, which were loosely associated with two waves of hepatocyte proliferation observed at 2 and 7 days after the surgery. Focusing on genes implicated in regulation of the progenitor cell activity, we came across slight increases in expression levels for Sox9 and two genes encoding tumor necrosis factor-like cytokine TWEAK (Tnfsf12) and its receptor Fn14 (Tnfrsf12a). At the same time, no increase in numbers of cytokeratin 19-positive (CK19⁺) cells was observed in periportal areas, and no CK19⁺ cells were found in hepatic plates. Since CK19 is thought to be a specific marker of both cholangiocytes and the hepatic progenitor cells, the data indicate a lack of activation of the resident progenitor cells during recovery of hepatic tissue after 80% subtotal hepatectomy. Thus, proliferation of hepatocytes invariably makes the major contribution to the hepatic tissue recovery, although in the cases of subtotal loss this contribution is distinctively modulated. In particular, induction of Sox9 and TWEAK/Fn14 regulatory pathways, conventionally attributed to progenitor cell activation, may incidentally stimulate mitotic activity of hepatocytes.</p></div

A set of markers with expression downregulated in the residual liver tissue.

<p>(<b>A</b>) Expression levels for <i>ang</i>, <i>vegf</i>, and <i>sdfa</i> (respectively <b>A</b>, <b>B</b>, and <b>C</b>) are plotted against time elapsed after the. The data are represented as mean values ± SEM with asterisks indicating statistical significance of differences (as compared to respective controls; <i>p</i><0.05).</p

A set of later markers with expression upregulated in the residual liver tissue at 5 to 10 days after subtotal hepatectomy.

<p>Expression levels for <i>il1b</i>, <i>tnfa</i>, <i>hgf</i>, and <i>tweak</i> (respectively <b>A</b>, <b>B</b>, <b>C</b>, and <b>D</b>) are plotted against time elapsed after the surgery. The data are represented as mean values ± SEM with asterisks indicating statistical significance of differences (as compared to respective controls; <i>p</i><0.05).</p

A set of earlier markers with expression upregulated in the residual liver tissue at 5 to 10 days after subtotal hepatectomy.

<p>Expression levels for <i>il6</i>, <i>il10</i>, <i>iNOs</i>, <i>mmp9</i>, <i>fgf2</i>, <i>tgfb</i>, <i>sox9</i>, and <i>fn14</i> (respectively <b>A</b>, <b>B</b>, <b>C</b>, <b>D</b>, <b>E</b>, <b>F</b>, <b>G</b>, and <b>H</b>) are plotted against time elapsed after the surgery. The data are represented as mean values ± SEM with asterisks indicating statistical significance of differences (as compared to respective controls; <i>p</i><0.05).</p

PCR primer structures and targets definition.

<p>PCR primer structures and targets definition.</p

Expression of CK19 and Sox9 proteins in the residual liver tissue after subtotal hepatectomy.

<p>(<b>А</b>) Relative CK19⁺ cell counts for sham operated liver in comparison with residual livers at different stages of regeneration measured by time elapsed after the surgery. (<b>B</b>) The diagram shows CK19⁺ index changing in the course of regeneration. (<b>С</b>) Evaluation of CK19 protein expression by western blot followed by quantitative densitometry. (<b>D</b>) Sox9⁺ cells in the sham operated liver tissue and in the residual livers at different stages of regeneration. Whereas Sox9 protein is found exclusively in the nuclei of cholangiocytes in the sham operated rat livers, it is expressed in the nuclei of hepatocytes after 80% subtotal resection. (<b>E</b>) The diagram shows Sox9⁺ index changing in the course of regeneration (<b>F</b>). Evaluation of Sox9 protein expression by western blot followed by quantitative densitometry. Exp–operated rats, SO—sham operated rats. The data are represented as mean values ± SEM with asterisks indicating statistical significance of differences (as compared to the control; <i>p</i><0.05).</p

Recovery rates of liver after 80% subtotal hepatectomy in rats.

<p>(<b>A</b>) Overall view of gross specimens in the intact state and at different days after the surgery; the plot shows recovery of the initial organ mass as measured at different days after the surgery and compared to the sham operated controls. (<b>B</b>) Serum concentrations of ALT and albumin in hepatectomized and sham operated rats. (<b>C</b>) Mitotic activity of hepatocytes in residual livers after 80% subtotal hepatectomy plotted against time elapsed after the surgery. No mitotic figures are observed in hepatocytes of the sham operated livers; the mitoses are induced by the surgery. Exp–operated rats, SO—sham operated rats. The data are represented as mean values ± SEM with asterisks indicating statistical significance of differences (as compared to the control; <i>p</i><0.05).</p

Dynamics of macrophage populations of the liver after subtotal hepatectomy in rats

Abstract Background In many clinical cases of extensive liver resection (e.g. due to malignancy), the residual portion is too small to maintain the body homeostasis. The resulting acute liver failure is associated with the compensatory growth inhibition, which is a typical manifestation of the ‘small for size’ liver syndrome. The study investigates possible causes of the delayed onset of hepatocyte proliferation after subtotal hepatectomy (80% liver resection) in rats. Results The data indicate that the growth inhibition correlates with delayed upregulation of the Tnf gene expression and low content of the corresponding Tnfα protein within the residual hepatic tissue. Considering the involvement of Tnf/Tnfα, the observed growth inhibition may be related to particular properties of liver macrophages – the resident Kupffer cells with CD68+CX1CR3−CD11b− phenotype. Conclusions The delayed onset of hepatocyte proliferation correlates with low levels of Tnfα in the residual hepatic tissue. The observed growth inhibition possibly reflects specific composition of macrophage population of the liver. It is entirely composed of embryonically-derived Kupffer cells, which express the ‘proregeneratory’ M2 macrophage-specific marker CD206 in the course of regeneration