Derek Whelan

Fibrin as a delivery system in wound healing tissue engineering applications

Fibrin is formed in the body upon initiation of the clotting cascade and is produced commercially for use as a tissue sealant and hemostasis device during surgical procedures. Experimentally fibrin is being increasingly used as a vector to deliver growth factors, cells, drugs and genes in tissue engineering applications mimicking aspects of the extra cellular matrix. Growth factors (GFs) are central to wound healing, inducing cell proliferation, migration and differentiation. Attempts have been made to augment wound healing with GFs, however widespread clinical use has been hindered in vivo due to their rapid metabolism within the body. Fibrin hydrogels protect GFs from rapid degradation and the composition of which can be altered to achieve their optimal release. This article reviews the use of fibrin for the delivery of GFs and details the various strategies that have evolved to alter the release rate so as to enhance the regenerative process, including bi-domain peptides, plasmin degradation sequences and heparin incorporation. This paper also reviews other recent advances in this field, such as dual delivery of cells and GF or sequential release of multiple GF.

Bone Marrow-Derived Mesenchymal Stem Cells Have Innate Procoagulant Activity and Cause Microvascular Obstruction Following Intracoronary Delivery: Amelioration by Antithrombin Therapy.

Mesenchymal stem cells (MSCs) are currently under investigation as tools to preserve cardiac structure and function following acute myocardial infarction (AMI). However, concerns have emerged regarding safety of acute intracoronary (IC) MSC delivery. This study aimed to characterize innate prothrombotic activity of MSC and identify means of its mitigation toward safe and efficacious therapeutic IC MSC delivery post‐AMI. Expression of the initiator of the coagulation cascade tissue factor (TF) on MSC was detected and quantified by immunofluorescence, FACS, and immunoblotting. MSC‐derived TF antigen was catalytically active and capable of supporting thrombin generation in vitro. Addition of MSCs to whole citrated blood enhanced platelet thrombus deposition on collagen at arterial shear, an effect abolished by heparin coadministration. In a porcine AMI model, IC infusion of 25 × 106 MSC during reperfusion was associated with a decrease in coronary flow reserve but not when coadministered with an antithrombin agent (heparin). Heparin reduced MSC‐associated thrombosis incorporating platelets and VWF within the microvasculature. Heparin‐assisted therapeutic MSC delivery also reduced apoptosis in the infarct border zone at 24 hours, significantly improved infarct size, left ventricular (LV) ejection fraction, LV volumes, wall motion, and attenuated histologic evidence of scar formation at 6 weeks post‐AMI. Heparin alone or heparin‐assisted fibroblast control cell delivery had no such effect. Procoagulant TF activity of therapeutic MSCs is associated with reductions in myocardial perfusion when delivered IC may be successfully managed by heparin coadministration. This study highlights an important mechanistic insight into safety concerns associated with therapeutic IC MSC delivery for AMI. Stem Cells 2015;33:2726–2737

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.

Synthetic chemically modified mrna-based delivery of cytoprotective factor promotes early cardiomyocyte survival post-acute myocardial infarction.

To extend the temporal window for cytoprotection in cardiomyocytes undergoing apoptosis after hypoxia and myocardial infarction (MI), a synthetic chemically modified mRNA (modRNA) was used to drive delivery of insulin-like growth factor-1 (IGF1) within the area at risk in an in vivo murine model of MI. Delivery of IGF1 modRNA, with a polyethylenimine-based nanoparticle, augmented secreted and cell-associated IGF1, promoting cardiomyocyte survival and abrogating cell apoptosis under hypoxia-induced apoptosis conditions. Translation of modRNA-IGF1 was sufficient to induce downstream increases in the levels of Akt and Erk phosphorylation. Downregulation of IGF1 specific miRNA-1 and -133 but not miR-145 expression was also confirmed. As a proof of concept, intramyocardial delivery of modRNA-IGF1 but not control modRNA-GFP significantly decreased the level of TUNEL positive cells, augmented Akt phosphorylation, and decreased caspase-9 activity within the infarct border zone 24 h post-MI. These findings demonstrate the potential for an extended cytoprotective effect of transient IGF1 driven by synthetic modRNA delivery.

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.

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.

Fibrin hydrogel as a novel delivery agent for local anesthetics: issues in demonstrating its efficacy at in vitro level

There is an identified need within the field of pain management to optimise the delivery of local anaesthetic agents as slow release formulations. Fibrin gel has shown huge potential for the local delivery of drugs including antibiotics, vasodilators, and chemotherapeutic agents however its potential for the delivery of local anaesthetic drugs has not, as yet, been fully investigated. Early randomized controlled trials have demonstrated efficacy, but to date, these have been relatively few and report the gross effect rather than specific kinetics. 1-3 The authors designed an in-vitro study aiming to evaluate sustained release of a local anesthetic agent from loaded fibrin gels over time. EDITOR, There is an identified need within the field of pain management to optimise the delivery of local anaesthetic agents as slow release formulations. Fibrin gel has shown huge potential for the local delivery of drugs including antibiotics, vasodilators, and chemotherapeutic agents however its potential for the delivery of local anaesthetic drugs has not, as yet, been fully investigated. Early randomized controlled trials have demonstrated efficacy, but to date, these have been relatively few and report the gross effect rather than specific kinetics. 1-3 The authors designed an in-vitro study aiming to evaluate sustained release of a local anesthetic agent from loaded fibrin gels over time. Some approximation was necessary in the methodology, as fibrin delivery of local anesthetics had not previously been investigated in vitro. Previous studies have addressed either local anaesthetic release from other delivery agents 4 or other drug release from fibrin gels. 5 Shemash et al. investigated controlled release of bupivacaine and ibuprofen from manufactured hybrid structures. 4 As part of their study bupivacaine loaded discshaped hybrids were immersed in vessels of phosphate buffered saline (PBS). At designated time intervals samples were withdrawn for analysis and the PBS medium replaced. Bupivacaine concentrations within the samples were assessed with high performance liquid chromatography (HPLC). HPLC has been validated as a rapid, Fibrin hydrogel as a novel delivery agent for local anesthetics: issues in demonstrating its efficacy at in vitro level SDRP Journal of Anesthesia and Surgery Letter to Editor ISSN: 2473-2184 DOI: 10.15436/JAS.2.2.3

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.