Evaluation of persistence and distribution of intra-dermally administered PKH26 labelled goat bone marrow derived mesenchymal stem cells in cutaneous wound healing model

The current study was designed to study the persistence and distribution of caprine bone marrow derived mesenchymal stem cells (cBM-MSCs) when administered intra-dermally in experimentally induced cutaneous wounds in rabbits. MSC’s from goat bone marrow were isolated and their differentiation potential towards adipogenic and osteogenic lineages were assayed in vitro. The isolated cells were phenotypically analysed using flow cytometry for the expression of MSC specific matrix receptors (CD73, CD105 and Stro-1) and absence of hematopoietic lineage markers. Further, these in vitro expanded MSCs were stained with PKH26 lipophilic cell membrane red fluorescent dye and prepared for transplantation into cutaneous wounds created on rabbits. Five, 2 cm linear full thickness skin incisions were created on either side of dorsal midline of New Zealand white rabbits (n = 4). Four wounds in each animal were implanted intra-dermally with PKH26 labelled cBM-MSCs suspended in 500 µl of Phosphate Buffer Saline (PBS). Fifth wound was injected with PBS alone and treated as negative control. The skin samples were collected from respective wounds on 3, 7, 10 and 14 days after the wound creation, and cryosections of 6 µM were made from it. Fluorescent microscopy of these cryosections showed that the PKH26 labelled transplanted cells and their daughter cells demonstrated a diffuse pattern of distribution initially and were later concentrated towards the wound edges and finally appeared to be engrafted with the newly developed skin tissues. The labelled cells were found retained in the wound bed throughout the period of 14 days of experimental study with a gradual decline in their intensity of red fluorescence probably due to the dye dilution as a result of multiple cell division. The retention of transplanted MSCs within the wound bed even after the complete wound healing suggests that in addition to their paracrine actions as already been reported, they may have direct involvement in various stages of intricate wound healing process which needs to be explored further.     

Evaluation of bone marrow derived mesenchymal stem cells for full-thickness wound healing in comparison to tissue engineered chitosan scaffold in rabbit

Background

Chronic wounds present a major challenge in modern medicine. Even under optimal conditions, the healing process may lead to scarring and fibrosis. The ability of mesenchymal stem cells (MSCs) to differentiate into other cell types makes these cells an attractive therapeutic tool for cell transplantation. Both tissue-engineered construct and MSC therapy are among the current wound healing procedures and potential care. Chitosan has been widely applied in tissue engineering because of its biocompatibility and biodegradability.

Fibroblasts/myofibroblasts that participate in cutaneous wound healing are not derived from circulating progenitor cells

Dermal fibroblasts/myofibroblasts involved in the wound healing are thought to originate from the resident fibroblast progenitors. To test the hypothesis of an extra dermal origin of the dermal fibroblasts/myofibroblasts, bone marrow (BM) transplantation and parabiosis experiments were initiated utilizing a collagen promoter green fluorescent protein (GFP) reporter transgene as a visible marker for dermal fibroblasts/myofibroblasts. BM transplantation experiments using BM from Col3.6GFPsapph transgenic mice showed no evidence that BM derived progenitors differentiated into dermal fibroblasts/myofibroblasts at the wound site. Rather the GFP positive cells (GFP+) observed at the wound site were not dermal fibroblasts/myofibroblasts but immune cells. These GFP+ cells were also detected in the lung and spleen. Furthermore, GFP+ fibroblasts were not detected in primary dermal fibroblast cultures initiated from BM chimeras. Using the same transgenic mice, parabiotic pairs were generated. One partner in the parabiosis carried a GFP expressing transgene while the other partner was a non‐transgenic C57BL/6 mouse. Similar to the BM transplantation experiments, GFP+ immune cells were detected in the wound of the non‐transgenic parabiont, however, GFP expressing dermal fibroblasts/myofibroblasts were not observed. Collectively, these data suggest that dermal fibroblast/myofibroblast progenitors do not readily circulate. The expression of the Col3.6GFPsapph in the hematopoietic cells confirmed that our methods were sensitive enough to detect Col3.6GFP expressing dermal fibroblasts derived from the peripheral circulation if they had originated in the BM. J. Cell. Physiol. 222: 703–712, 2010. © 2009 Wiley‐Liss, Inc.     

Transplantation with bone marrow stromal cells promotes wound healing under chemotherapy through altering phenotypes

Stem cell transplantation is a promising strategy for delayed wound healing caused by chemotherapy. However, the fate of stem cells under chemotherapy has not been fully elucidated. Herein we characterized human fetal bone marrow stromal cells (hBMSCs) during wound healing in mice treated with cyclophosphamide (CTX). The isolated hBMSCs expressed the phenotype of CD11b(low)/CD14(low)/CD34(low)/CD45(low)/CD29(high)/CD44(high)/CD90(high)/CD105(high)/CD146(high)/STRO-1(low). Following in vitro exposure to CTX, hBMSCs showed decreased cell growth in a dose- and time-dependent manner, accompanied by increased expressions of collagen-I/III, and CD31. After transplantation, wounds closed as early as 8 days and were positive for α-smooth muscle actin (α-SMA), implicating the enhanced re-epithelialization and wound contraction. Moreover, proliferating cell nuclear antigen (PCNA) and CD31 showed co-localization with α-SMA, suggesting the differentiation of hBMSCs into epithelial cells and myofibroblasts/fibroblasts. Taken together, our results indicate hBMSCs can accelerate wound healing under chemotherapy through altering their phenotypes.     

Participation of transfused bone marrow cells in reparative osteohistogenesis

The participation of skeletal tissue cell precursors in the repairing regeneration of bone tissue was studied. Bone marrow was taken from donor animals--mice of C57Bl/6-TgN(ACTbGFP) 1 Osb line (The Jackson Laboratory Bar Harbor ME USA line). Nucleated cell fraction was isolated by centrifugation on a density percoll gradient. Recipient mice C57Bl/6 line were irradiated by 7.0-7.5 Gr dose. Intravenous infusion of donor cells and osteoclasts of tibia was done after irradiation of recipient mice. Histological preparations of bone regenerate tissues were studied on 15, 30, and 60 days by confocal microscopy. Donor cells were found as skeletal tissue precursors into periost, endost, bone marrow, and as differentiated cells of newborn tissue of regenerate--osteoblasts, osteocytes, chondrocytes. The data obtained indicate that part of donor bone marrow cells are able to progressive differentiation under recipient bone fractures.     

Keratinocytes derived from embryonic stem cells induce wound healing in mice

The skin plays an important role in defending the body against the environment. Treatments for burns and skin injuries that use autologous or allogenic skin grafts derived from adult or embryonic stem cells are promising. Embryonic stem cells are candidates for regenerative and reparative medicine. We investigated the utility of keratinocyte-like cells, which are differentiated from mouse embryonic stem cells, for wound healing using a mouse surgical wound model. Mice were allocated to the following groups: experimental, in which dressing and differentiated cells were applied after the surgical wound was created; control, in which only the surgical wound was created; sham, in which only the dressing was applied after the surgical wound was created; and untreated animal controls with healthy skin. Biopsies were taken from each group on days 3, 5 and 7 after cell transfer. Samples were fixed in formalin, then stained with Masson’s trichrome and primary antibodies to interleukin-8 (IL-8), fibroblast growth factor-2 (FGF-2), monocyte chemoattractant protein-1 (MCP-1), collagen-1 and epidermal growth factor (EGF) using the indirect immunoperoxidase technique for light microscopy. Wound healing was faster in the experimental group compared to the sham and control groups. The experimental group exhibited increased expression of IL-8, FGF-2 and MCP-1 during early stages of wound healing (inflammation) and collagen-1 and EGF expression during late stages of wound healing (proliferation and remodeling). Keratinocytes derived from embryonic stem cells improved wound healing and influenced the wound healing stages.     

MicroRNA 在皮肤创伤愈合中的作用

皮肤创伤愈合过程是一个复杂而连续的过程,这一过程需要多种细胞、多种因子的参与,涉及细胞增殖、细胞分化、细胞运动、细胞黏附等多个细胞生物学过程。 MicroRNA（ miRNA）是一类高度保守的非编码RNA,它通过靶向结合信使RNA（ mRNA）并使其降解或抑制其翻译,实现转录后基因表达调控。 miRNA作为基因表达的重要调控分子,几乎参与了机体所有的生理和病理过程。除了在皮肤发育中发挥重要的作用,还参与多种皮肤病、皮肤癌和皮肤创伤愈合过程的调节。主要总结了miRNA调控皮肤创伤愈合的研究进展。     

Participation of the lacrimal glands in wound healing processes

A hypothesis is advanced that nociceptive stimulation activates the functional system of wound healing. Experiments were made to investigate whether the lacrimal glands, which are activated due to pain, are involved in the system of healing. Rat experiments have demonstrated that nociceptive stimulation of the lacrimal glands accelerates the repair of cutaneous wounds by 26 to 30%. Lacrimectomy accompanied by distension of wound rims (up to 26% by the 3d-4th hour) inhibits the rate of healing by 18 to 20%. Intraperitoneal injection of a lacrimal gland extract activates healing in lacrimectomized rats. The data obtained suggest that the lacrimal glands are involved in wound healing.     

Effects of genistein on early-stage cutaneous wound healing

Wound healing occurs in three sequential phases: hemostasis and inflammation, proliferation, and remodeling. Inflammation, the earliest phase, is considered a critical period for wound healing because immune cells remove damaged tissues, foreign debris, and remaining dead tissue. Wound healing would be delayed without inflammation, and this phase is affected by antioxidation capacity. Therefore, we hypothesized that genistein, which has an antioxidant effect, might modulate the wound healing process by altering the inflammatory response. After three days of acclimation, mice were divided into three groups: control, 0.025% genistein, and 0.1% genistein. After two weeks of an experimental diet, skin wounds were induced. Wounded skin areas were imaged, and the healing rate calculated. To measure lipid peroxidation, antioxidant enzyme expression and activity, and pro-inflammatory cytokine expression, skin and liver tissues were harvested at 12, 24, 48, and 72 h. Genistein did not affect body weight. The rate of wound closure in mice fed genistein was significantly faster than in the control group during the early stage of wound healing, especially in first three days. Cu, Zn-SOD and Mn-SOD expression in wound skin tissue in the 0.1% genistein group was lower than in the control group. However, CAT expression did not differ among groups. We also found that genistein modulated NF-κB and TNF-α expression during the early stage of wound healing. The genistein group had significantly lower hepatic lipid peroxidation and higher SOD, CAT, and GPx activities than the control group. These results suggest that genistein supplementation reduces oxidative stress by increasing antioxidant capacity and modulating proinflammatory cytokine expression during the early stage of wound healing.     

Exercise accelerates cutaneous wound healing and decreases wound inflammation in aged mice

The purpose of this study was to determine the effect of exercise on wound healing and inflammation in young (3 mo) and old (18 mo) female BALB/cByJ mice. Mice were assigned to either exercise or sedentary control (control) groups. The exercise group mice were run on a motorized treadmill at a moderate intensity 30 min/day for 8 days. All mice were given four full-thickness dermal wounds, and the rate of wound closure was assessed daily for 10 days. Four months later, the aged mice were rerandomized to treatment, wounded again in different locations, and wounds were harvested at 1, 3, or 5 days postwounding. Wound tissue was analyzed for IL-1beta, IL-6, keratinocyte chemoattractant (KC), monocyte chemoattractant protein-1 (MCP-1), and TNF-alpha protein. Myeloperoxidase (MPO) activity and F4/80 mRNA were assessed as an indirect measure of neutrophil and macrophage content, respectively. There was a trend (P = 0.10) for exercise to reduce wound size in young mice, and exercise significantly (P < 0.05) decreased wound size in old mice. TNF-alpha, KC, and MCP-1 were significantly (P < 0.05) lower in wounds from exercised old mice compared with control. No group differences were found for wound IL-1beta or IL-6, MPO activity, or F4/80 mRNA. Our data suggest that exercise accelerates the wound healing process in old mice. This improved healing response in the old mice may be the result of an exercise-induced anti-inflammatory response in the wound.     

Delayed re-epithelialization in periostin-deficient mice during cutaneous wound healing

Background

Matricellular proteins, including periostin, are important for tissue regeneration.

Methods and Findings

Presently we investigated the function of periostin in cutaneous wound healing by using periostin-deficient (−/−) mice. Periostin mRNA was expressed in both the epidermis and hair follicles, and periostin protein was located at the basement membrane in the hair follicles together with fibronectin and laminin γ2. Periostin was associated with laminin γ2, and this association enhanced the proteolytic cleavage of the laminin γ2 long form to produce its short form. To address the role of periostin in wound healing, we employed a wound healing model using WT and periostin−/− mice and the scratch wound assay in vitro. We found that the wound closure was delayed in the periostin−/− mice coupled with a delay in re-epithelialization and with reduced proliferation of keratinocytes. Furthermore, keratinocyte proliferation was enhanced in periostin-overexpressing HaCaT cells along with up-regulation of phosphorylated NF-κB.

Conclusion

These results indicate that periostin was essential for keratinocyte proliferation for re-epithelialization during cutaneous wound healing.     

Antler stem cells and their potential in wound healing and bone regeneration

Compared to other vertebrates, the regenerative capacity of appendages in mammals is very limited. Deer antlers are an exception and can fully regenerate annually in postnatal mammals. This process is initiated by the antler stem cells (AnSCs). AnSCs can be divided into three types: (1) Antlerogenic periosteum cells (for initial pedicle and first antler formation); (2) Pedicle periosteum cells (for annual antler regeneration); and (3) Reserve mesenchyme cells (RMCs) (for rapid antler growth). Previous studies have demonstrated that AnSCs express both classic mesenchymal stem cells (MSCs) and embryonic stem cells (ESCs), and are able to differentiate into multiple cell types in vitro. Thus, AnSCs were defined as MSCs, but with partial ESC attributes. Near-perfect generative wound healing can naturally occur in deer, and wound healing can be achieved by the direct injection of AnSCs or topical application of conditioned medium of AnSCs in rats. In addition, in rabbits, the use of both implants with AnSCs and cell-free preparations derived from AnSCs can stimulate osteogenesis and repair defects of bone. A more comprehensive understanding of AnSCs will lay the foundation for developing an effective clinical therapy for wound healing and bone repair.     

Investigating a simple model of cutaneous wound healing angiogenesis

A simple model of wound healing angiogenesis is presented, and investigated using numerical and asymptotic techniques. The model captures many key qualitative features of the wound healing angiogenic response, such as the propagation of a structural unit into the wound centre. A detailed perturbative study is pursued, and is shown to capture all features of the model. This enables one to show that the level of the angiogenic response predicted by the model is governed to a good approximation by a small number of parameter groupings. Further investigation leads to predictions concerning how one should select between potential optimal means of stimulating cell proliferation in order to increase the level of the angiogenic response.     

Secretome analysis of human bone marrow derived mesenchymal stromal cells

As an essential cellular component of the bone marrow (BM) microenvironment mesenchymal stromal cells (MSC) play a pivotal role for the physiological regulation of hematopoiesis, in particular through the secretion of cytokines and chemokines. Mass spectrometry (MS) facilitates the identification and quantification of a large amount of secreted proteins (secretome), but can be hampered by the false-positive identification of contaminating proteins released from dead cells or derived from cell medium. To reduce the likelihood of contaminations we applied an approach combining secretome and proteome analysis to characterize the physiological secretome of BM derived human MSC. Our analysis revealed a secretome consisting of 315 proteins. Pathway analyses of these proteins revealed a high abundance of proteins related to cell growth and/or maintenance, signal transduction and cell communication thereby representing key biological functions of BM derived MSC on protein level. Within the MSC secretome we identified several cytokines and growth factors such as VEGFC, TGF-β1, TGF-β2 and GDF6 which are known to be involved in the physiological regulation of hematopoiesis. By comparing the peptide patterns of secretomes and cell lysates 17 proteins were identified as candidates for proteolytic processing. Taken together, our combined MS work-flow reduced the likelihood of contaminations and enabled us to carve out a specific overview about the composition of the secretome from human BM derived MSC. This methodological approach and the specific secretome signature of BM derived MSC may serve as basis for future comparative analyses of the interplay of MSC and HSPC in patients with hematological malignancies.