Mesenchymal stromal cell derived CCL2 is required for accelerated wound healing.

Mesenchymal stromal cells (MSC) have immunomodulatory effects impacting macrophages, promoting polarisation towards a reparative phenotype. CCL2 is a potent cytokine involved in the recruitment of macrophages. We hypothesised that MSC derived CCL2 may be involved in the MSC therapeutic effect by facilitating macrophage repolarisation. To further delineate this mechanism, MSC isolated from CCL2 deficient mice (MSC-KO) were applied to excisional wounds in wild-type (WT) mice. CCL2 deficiency in MSC completely abrogated the therapeutic response compared to MSC-WT. MSC-KO were unable to repolarise macrophages to the same extent as WT and this was accompanied by a reduced angiogenesis and re-epithelialisation of the wounds at day 10. This study demonstrates that MSC derived CCL2 is required for MSC induced accelerated wound healing. The role of CCL2 in the interaction between MSC and Macrophages has not been previously demonstrated in accelerated wound healing. CCL2 has a potent effect on the ability to reduce the inflammatory response through local recruitment of macrophages. This research highlights CCL2 as a possible target for augmentation of MSC therapy to enhance therapeutic potential.

The Use of Thromboelastography to Measure the Influence Inclusion of a Local Anesthetic Agent has on the Mechanical and Kinetic Properties of Fibrin.

CONTEXT: Delivery of slow-release local anesthesia has considerable potential for postoperative analgesia. Fibrin gel has shown huge potential for drug delivery, but has not been fully investigated for the delivery of local anesthetics nor has whether incorporation of anesthetic drugs into fibrin alters its mechanical properties. AIMS: This study aimed to evaluate the effects of bupivacaine inclusion on the mechanical and kinetic properties of fibrin as measured by thromboelastography (TEG). MATERIALS AND METHODS: Serial dilutions of fibrinogen with thrombin were tested with TEG to identify the optimal concentrations to give reproducible results. Following this, fibrinogen samples diluted with bupivacaine 0.5% in place of normal saline (also 1:20 dilution) were added to thrombin to assess what influence this had on clot strength and kinetics as measured by TEG values (with R, K, and α angle relating to clot kinetics and MA and G (or shear elastic modulus strength) relating to clot strength). RESULTS: The mean values yielded for R were higher and lower for α angle, suggesting that the inclusion of bupivacaine produced a fibrin clot at a slower rate. The values for MA and G were both lower when bupivacaine was included, suggesting inclusion of the local anaesthetic also resulted in a fibrin clot of inferior strength. These results were not statistically significant. CONCLUSION: Although TEG failed to consistently measure these properties, the results suggest that inclusion of local anesthetic affects the clotting process of fibrin, potentially interfering with its ability to function as a sealant, adhesive, or hemostat.

Selective M2 Macrophage Depletion Leads to Prolonged Inflammation in Surgical Wounds.

BACKGROUND: A prolonged inflammatory phase is seen in aberrant wound healing and in chronic wounds. Macrophages are central to wound healing. Distinct macrophage subtypes have differing roles both in initial inflammation and in later tissue repair. Broadly, these cells can be divided into M1 and M2 macrophages. M2 macrophage proliferation and differentiation is regulated by colony-stimulating factor 1 (CSF-1) signalling and can be blocked by GW2580, a competitive cFMS kinase inhibitor, thereby allowing for analysis of the effect of M2 blockade on progression of surgical wounds. MATERIALS AND METHODS: Macrophage Fas-induced apoptosis (MaFIA) transgenic mice with a macrophage-specific promoter used to express green fluorescent protein (GFP) were used to allow for cell tracking. The animals were treated by oral gavage with GW2580. Surgical wounds were created and harvested after 2 weeks for analysis. RESULTS: GW2580-treated mice had significantly more GFP+ cells in the surgical scar than vehicle-treated animals (GW2580, 68.0 ± 3.1%; vehicle, 42.8 ± 1.7%; p < 0.001), and GW2580 treatment depleted CD206+ M2 macrophages in the scar (GW2580, 1.4%; vehicle, 19.3%; p < 0.001). Treated animals showed significantly higher numbers of neutrophils (vehicle, 18.0%; GW2580, 51.3%; p < 0.01) and M1 macrophages (vehicle, 3.8%; GW2580, 12.8%; p < 0.01) in the scar compared to vehicle-treated animals. The total collagen content in the area of the scar was decreased in animals treated with GW2580 as compared to those treated with vehicle alone (GW2580, 67.1%; vehicle, 79.9%; p < 0.005). CONCLUSIONS: Depletion of M2 macrophages in surgical wounds via CSF-1 signalling blockade leads to persistent inflammation, with an increase in neutrophils and M1 macrophages and attenuated collagen deposition.

Novel methods of local anesthetic delivery in the perioperative and postoperative setting-potential for fibrin hydrogel delivery.

The benefits of high-quality postoperative analgesia are well documented and include earlier mobilization, fewer respiratory and cardiovascular complications, and shorter hospital stay. Local anesthesia-based acute pain regimens are at worst equal to and at best superior to opiate-based regimens from the perspective of analgesia. A multimodal approach limiting opioids by combining with local anesthetics has additional beneficial effect on outcomes such as nausea and vomiting, pruritus, gastrointestinal function, respiratory complications, and neutrophil function. Wound catheters providing continuous infiltration of local anesthetics offer a rational approach to effective perioperative analgesia, but their use is limited by a short duration of action. There is an identified need for further methods to optimize longer-acting delivery of these agents. This article reviews current and evolving longer-acting techniques and their limitations with particular focus on the potential advantages of a fibrin hydrogel-based system.

Preclinical evaluation of an endoscopic electroporation system.

BACKGROUND AND STUDY AIMS: Targeted delivery of specific chemotherapeutic drugs into tumors can be achieved by delivering electrical pulses directly to the tumor tissue. This causes a transient formation of pores in the cell membrane that enables passive diffusion of normally impermeant drugs. A novel device has been developed to enable the endoscopic delivery of this tumor permeabilizing treatment. The aim of the preclinical studies described here was to investigate the efficacy and safety of this nonthermal ablation system in the treatment of gastrointestinal cancer models. METHODS: Murine, porcine, and canine gastrointestinal tumors and tissues were used to assess the efficacy and safety of electroporation delivered through the special device in combination with bleomycin. Tumor cell death, volume, and overall survival were recorded. RESULTS: Murine tumors treated with electrochemotherapy showed excellent responses, with cell death being induced rapidly, mainly via an apoptotic-type mechanism. Use of the system in canine gastrointestinal cancers demonstrated successful local endoluminal tumor resolution, with no safety or adverse effects noted. CONCLUSIONS: Electroporation via the new device in combination with bleomycin offers a nonthermal tumor ablative approach, and presents clinicians with a new option for the management of gastrointestinal cancers.

A Novel Selectable Islet 1 Positive Progenitor Cell Reprogrammed to Expandable and Functional Smooth Muscle Cells.

Disorders affecting smooth muscle structure/function may require technologies that can generate large scale, differentiated and contractile smooth muscle cells (SMC) suitable for cell therapy. To date no clonal precursor population that provides large numbers of differentiated SMC in culture has been identified in a rodent. Identification of such cells may also enhance insight into progenitor cell fate decisions and the relationship between smooth muscle precursors and disease states that implicate differentiated SMC. In this study, we used classic clonal expansion techniques to identify novel self-renewing Islet 1 (Isl-1) positive primitive progenitor cells (PPC) within rat bone marrow that exhibited canonical stem cell markers and preferential differentiation towards a smooth muscle-like fate. We subsequently used molecular tagging to select Isl-1 positive clonal populations from expanded and de novo marrow cell populations. We refer to these previously undescribed cells as the PPC given its stem cell marker profile, and robust self-renewal capacity. PPC could be directly converted into induced smooth muscle cells (iSMC) using single transcription factor (Kruppel-like factor 4) knockdown or transactivator (myocardin) overexpression in contrast to three control cells (HEK 293, endothelial cells and mesenchymal stem cells) where such induction was not possible. iSMC exhibited immuno- and cytoskeletal-phenotype, calcium signaling profile and contractile responses similar to bona fide SMC. Passaged iSMC could be expanded to a scale sufficient for large scale tissue replacement. PPC and reprogramed iSMC so derived may offer future opportunities to investigate molecular, structure/function and cell-based replacement therapy approaches to diverse cardiovascular, respiratory, gastrointestinal, and genitourinary diseases that have as their basis smooth muscle cell functional aberrancy or numerical loss. Stem Cells 2016;34:1354-1368.

Intraperitoneal co-administration of low dose urethane with xylazine and ketamine for extended duration of surgical anesthesia in rats.

Procedures involving complex surgical techniques in rats, such as placement of abdominal aortic graft require extended duration of surgical anesthesia, which often can be achieved by repeated administrations of xylazine-ketamine combination. However such repeated anesthetic administration, in addition to being technically challenging, may be associated with potential adverse events due to cumulative effects of anesthesia. We report here the feasibility of using urethane at low dose (~1/10 the recommended anesthetic dose) in combination with a xylazine-ketamine mix to achieve an extended duration of surgical anesthesia in rats. The anesthesia induction phase was quick and smooth with an optimal phase of surgical anesthesia achieved for up to 90 minutes, which was significantly higher compared to that achieved with use of only xylazine-ketamine combination. The rectal temperature, heart rate and respiratory rate were within the physiological range with an uneventful recovery phase. Post surgery the rats were followed up to 3 months without any evidence of tumor or any other adverse effects related to the use of the urethane anesthetic combination. We conclude that low dose urethane can be effectively used in combination with xylazine and ketamine to achieve extended duration of surgical anesthesia up to 90 minutes in rats.

Allogeneic mesenchymal stem cells, but not culture modified monocytes, improve burn wound healing.

The use of cell therapy to improve burn wound healing is limited as a validated cell source is not rapidly available after injury. Progenitor cells have shown potential to drive the intrinsic wound regeneration. Two sources of cells, allogeneic mesenchymal stem cells (MSC) and autologous culture modified monocytes (CMM), were assessed for their ability to influence burn wound healing. Both could be widely available shortly after injury. Cells were delivered in a fibrin matrix following contact burns in a porcine burns model. Application of MSC significantly decreased the area of unhealed burn compared to CMM or delivery matrix alone (6% MSC, 27% CMM, 24% Matrix, p<0.001). MSC treated wounds showed histological evidence of improved wound healing with increased collagen content (MSC 49%, CMM 42%, p<0.01), increased epidermal area (MSC 8.8%, CMM 6.1%, p<0.01) and dermal thickness (MSC 1108 µm, CMM 1007 µm, p<0.01) compared to CMM treated wounds. Labelled MSC and CMM were identified in the wounds after 2 weeks by immunohistochemistry and FACS. A single application of allogeneic MSC improves the rate of burn wound healing and improves the histological appearance of the burn wound. These cells show potential as a cell therapy that is rapidly available following burn.

Analysis of attitudes toward the source of progenitor cells in tissue-engineered products for use in burns compared with other disease states.

The first trials using progenitor cells to improve burn wound healing are beginning. However, there remains a paucity of data on patients' opinions of the source of stem cells. In this study, 279 patients attending plastic surgery/burns outpatient and medical outpatient clinics were questioned to assess willingness to accept a tissue-engineered skin product derived from a variety of sources. Levels of acceptance for the use of progenitor cells derived from these sources for treatment across a range of disease states (burns, Parkinson's disease, diabetes, and for cosmetic use) were also assessed. Overall, 80% of those questioned would accept a tissue-engineered product. Autologous cells were the preferred choice of cells (acute burns 94%, diabetes 95%, Parkinson's 93.9%). Allogeneic cells were still widely accepted (acute burns 67%, diabetes 66.7%, Parkinson's 69.2%). There was no difference observed between plastic surgical patients and medical patients in acceptance of cell therapy for burns, Parkinson's disease, or diabetes. There is good potential acceptance for the use of both autologous and allogeneic cells for the treatment of acute burns and burns' scarring as well as in diabetes and Parkinson's disease. Disease state does not appear to influence overall acceptability and choice of cells.

Deficiency of CX3CR1 delays burn wound healing and is associated with reduced myeloid cell recruitment and decreased sub-dermal angiogenesis.

The development of a good blood supply is a key step in burn wound healing and appears to be regulated in part by myeloid cells. CX3CR1 positive cells have recently been identified as myeloid cells with a potential role in angiogenesis. The role of functional CX3CR1 system in burn wound healing is not previously investigated. A 2% contact burn was induced in CX3CR1(+/gfp) and CX3CR1(gfp/gfp) mice. These transgenic mice facilitate the tracking of CX3CR1 cells (CX3CR1(+/gfp)) and allow evaluation of the consequence of CX3CR1 functional knockout (CX3CR1(gfp/gfp)) on burn wound healing. The progression of wound healing was monitored before tissue was harvested and analyzed at day 6 and day 12 for migration of CX3CR1 cells into burn wound. Deficiency of a functional CX3CR1 system resulted in decreased recruitment of CX3CR1 positive cells into the burn wound associated with decreased myeloid cell recruitment (p<0.001) and reduced maintenance of new vessels (p<0.001). Burn wound healing was prolonged (p<0.05). Our study is the first to establish a role for CX3CR1 in burn wound healing which is associated with sub-dermal angiogenesis. This chemokine receptor pathway may be attractive for therapeutic manipulation as it could increase sub dermal angiogenesis and thereby improve time to healing.