Thymosin β4 has a major role in dermal burn wound healing that involves actin cytoskeletal remodelling via heat‐shock protein 70

Rapid vascular remodelling of damaged dermal tissue is required to heal burn wounds. Thymosin β4 (Tβ4) is a growth factor that has been shown to promote angiogenesis and dermal wound repair. However, the underlying mechanisms based on Tβ4 function have not yet been fully investigated. In the present study, we investigated how Tβ4 improves dermal burn wound healing via actin cytoskeletal remodelling and the action of heat‐shock proteins (HSPs), which are a vital set of chaperone proteins that respond to heat shock. Our in vitro results achieved with the use of human umbilical vein endothelial cells (HUVECs) revealed a possible signal between Tβ4 and HSP70. Moreover, we confirmed that remodelling of filamentous actin (F‐actin) was regulated by Tβ4‐induced HSP70 in HUVECs. Based on these in vitro results, we confirmed the healing effects of Tβ4 in an adapted dermal burn wound in vivo model. Tβ4 improved wound‐healing markers, such as wound closure and vascularization. Moreover, Tβ4 maintained the long‐term expression of HSP70, which is associated with F‐actin regulation during the wound‐healing period. These results suggest that an association between Tβ4 and HSP70 is responsible for the healing of burn wounds, and that this association may regulate F‐actin remodelling. Copyright © 2015 John Wiley & Sons, Ltd.     

Platelet‐rich fibrin‐based matrices to improve angiogenesis in an in vitro co‐culture model for bone tissue engineering

In the context of prevascularization strategies for tissue‐engineering purposes, co‐culture systems consisting of outgrowth endothelial cells (OECs) and primary osteoblasts (pOBs) have been established as a promising in vitro tool to study regeneration mechanisms and to identify factors that might positively influence repair processes such as wound healing or angiogenesis. The development of autologous injectable platelet‐rich fibrin (PRF), which can be generated from peripheral blood in a minimal invasive procedure, fulfils several requirements for clinically applicable cell‐based tissue‐engineering strategies. During this study, the established co‐culture system of OECs and pOBs was mixed with injectable PRF and was cultivated in vitro for 24 h or 7 days. The aim of this study was to analyse whether PRF might have a positive effect on wound healing processes and angiogenic activation of OECs in the co‐culture with regard to proinflammatory factors, adhesion molecules and proangiogenic growth factor expression. Histological cell detection revealed the formation of lumina and microvessel‐like structures in the PRF/co‐culture complexes after 7 days of complex cultivation. Interestingly, the angiogenic activation of OECs was accompanied by an upregulation of wound healing‐associated factors, as well as by a higher expression of the proangiogenic factor vascular endothelial growth factor, which was evaluated both on the mRNA level as well as on the protein level. Thus, PRF might positively influence wound healing processes, in particular angiogenesis, in the in vitro co‐culture, making autologous PRF‐based matrices a beneficial therapeutic tool for tissue‐engineering purposes by simply profiting from the PRF, which contains blood plasma, platelets and leukocytes.     

Efficacy and safety of a fibrin sealant for adherence of autologous skin grafts to burn wounds: results of a phase 3 clinical study.

The objective of this phase 3, multicentered, prospective, randomized, evaluator-blinded, clinical study was to compare skin graft adherence utilizing a fibrin sealant containing 4 IU/ml thrombin (FS 4IU VH S/D [FS 4IU VH S/D will be marketed under the trade name ARTISS upon licensure in the United States]) to graft adherence utilizing staples in burn patients requiring wound excision and skin grafting. FS 4IU VH S/D was compared with staples in 138 patients. Patients had burn wounds measuring < or =40% of total body surface area with two comparable test sites measuring between 1 and 4% total body surface area each. Wound closure at day 28 was assessed using test site planimetry and review of day 28 photographs by three independent blinded evaluators (primary endpoint analysis). Secondary efficacy measures included hematoma/seroma on day 1, engraftment on day 5, and wound closure on day 14. Investigator and patient-reported outcomes were also assessed. The proportion of test sites with complete wound closure at day 28 was 70.3% in FS 4IU VH S/D treated sites and 65.8% in stapled sites, as assessed by planimetry. Blinded review of day 28 photographs confirmed that the rate of complete wound closure was similar between the two treatments, although the overall assessed rates of closure were lower than those determined by planimetry: FS 4IU VH S/D (43.3%) and staples (37.0%). The lower limit of the 97.5% confidence interval of the difference between FS 4IU VH S/D and staples was -0.029, which is above the predefined noninferiority margin of -0.1. Therefore, FS 4IU VH S/D is at least as efficacious as staples at the 97.5% one-sided level for complete wound closure by day 28. Hematoma/seroma on day 1 occurred at significantly (P < .0001) fewer FS 4IU VH S/D-treated sites (29.7% [95% CI 22.2-38.1%]) compared with stapled sites (62.3% [95% CI 53.7-70.4%]). Engraftment on day 5 was deemed to be 100% in 62.3% (95% CI 53.7-70.4%) of the FS 4IU VH S/D-treated sites and 55.1% (95% CI 46.4-63.5%) of the stapled sites (P = .0890). Complete wound closure by day 14 occurred in 48.8% (95% CI 39.9-57.8%) of the FS 4IU VH S/D treated sites and 42.6% (95% CI 34.0-51.6%) of the stapled sites (P = .2299). FS 4IU VH S/D scored significantly better than staples for all investigator-assessed outcomes, namely quality of graft adherence (P < .0001), preference for method of fixation (P < .0001), satisfaction with graft fixation (P < .0001), and overall quality of healing (P < .0001). Likewise, FS 4IU VH S/D scored significantly better than staples for all patient-assessed outcomes, namely anxiety about pain (P < .0001) and treatment preference (P <.0001). The safety profile of FS 4IU VH S/D was excellent as indicated by the lack of any related serious adverse experiences. These findings demonstrate that FS 4IU VH S/D is safe and effective for attachment of skin grafts, with outcomes at least as good as or better than staple fixation.     

Acceleration of skin regeneration in full‐thickness burns by incorporation of bFGF‐loaded alginate microspheres into a CMCS–PVA hydrogel

Two important issues in skin tissue engineering are the vascularization and regeneration of the dermis. Basic fibroblast growth factor (bFGF) is known to promote angiogenesis and accelerate wound healing. Direct delivery of bFGF to the wound area, however, would lead to a loss of bioactivity. To this end, bFGF‐loaded alginate microspheres (Ms) were fabricated and incorporated into carboxymethyl chitosan (CMCS)–poly(vinyl alcohol) (PVA) to form a composite hydrogel. Scanning electron microscopy (SEM) results indicated that the incorporation of Ms does not significantly affect the inner structure of CMCS–PVA. In an in vitro study, the release of bFGF from Ms–CMCS–PVA in a sustained manner retained higher bioactivity over a 2‐week period. Full‐thickness burn wounds were created in the dorsal area of rats for in vivo evaluation of skin regeneration treated with CMCS–PVA hydrogel, with and without bFGF. Compared with the control, CMCS–PVA and bFGF–CMCS–PVA groups, the bFGF/Ms–CMCS–PVA group revealed significantly faster wound recovery rates, with re‐epithelialization and regeneration of the dermis. Moreover, the bFGF/Ms–CMCS–PVA group had the highest density of newly formed and mature blood vessels during the 2 mweek treatment period. The ability of the bFGF/Ms–CMCS–PVA hydrogel to accelerate wound healing in a full‐thickness burn model suggests its potential for use in dermal tissue regeneration. Copyright © 2015 John Wiley & Sons, Ltd.     

Collagen‐cellulose nanocrystal scaffolds containing curcumin‐loaded microspheres on infected full‐thickness burns repair

Burn infection is a serious problem that delays wound healing and leads to death. Curcumin (Cur) has been shown to exhibit antioxidant, anti‐inflammatory, antimicrobial and anticarcinogenic activity. However, its instability, extremely low aqueous solubility and bioavailability in physiological fluids may make it difficult to maintain local Cur concentrations above the minimum inhibitory concentration for burn infection treatment. The objective of this study was to construct complexes of Cur/gelatin microspheres (GMs) and porous collagen (Coll)‐cellulose nanocrystals (CNCs) composite scaffolds for full‐thickness burn infection treatment. The Cur/GMs/Coll‐CNCs scaffolds had high porosity, available pore size, and a long and sustained Cur release profile. Furthermore, the composite scaffold exhibited remarkably strong antibacterial activity. Hence, we evaluated the wound‐healing effects and antibacterial properties of Cur/GMs/Coll‐CNCs scaffolds in a rat full‐thickness burn infection model. The Cur/GMs/Coll‐CNCs scaffold was able to prevent not only local inflammation but also accelerated dermis regeneration. Thus, we conclude that Cur/GMs/Coll‐CNCs scaffolds can act as an effective dermal regeneration template for full‐thickness burn wound infection healing in rats models. Copyright © 2017 John Wiley & Sons, Ltd.     

Release of platelet-derived growth factors and proliferation of fibroblasts in the releasates from platelets stored in the liquid state at 22 degrees C after stimulation with agonists

BACKGROUND: Fresh platelet (PLT)-rich plasma (PRP) treated with thrombin plus calcium chloride (CaCl(2)) is used to prepare a PLT gel to promote hemostasis and wound healing in a variety of surgical procedures. The effects of various agonists on stimulating the release of growth factors from liquid-preserved PLTs and the effects of the PLT releasate on the growth of fibroblasts in tissue culture were investigated. STUDY DESIGN AND METHODS: Plateletpheresis PLTs stored at 22 degrees C as high-yield PLTs for 3 to 6 days or outdated PLTs for 9 days were treated with agonists to assess release of platelet-derived growth factor (PDGF) AA, PDGF AB, PDGF BB, transforming growth factor-beta1 (TGF-beta1), and osteocalcin and the proliferation of fibroblasts treated with the PLT releasates in tissue culture. RESULTS: All treatments except for CaCl(2) alone and zeolite-CaCl(2) produced significant increases in PDGF AA compared to PRP. Thrombin-CaCl(2) produced significant increases in PDGF BB. Treatment by all the agonists produced similar increases in PDGF AB. TGF-beta1 and osteocalcin levels after treatment were similar to those in PRP. PRP releasate before and after stimulation with different agonists increased proliferation of fibroblasts in tissue culture. CONCLUSION: High-yield and outdated liquid-preserved PLTs released PDGF AA, AB, and BB but not TGF-beta1 or osteocalcin. The releasate from untreated PRP stimulated the proliferation of fibroblasts in tissue culture similar to the releasates from PRP treated with the different agonists. Further studies are needed to assess whether or not high-yield and outdated PLTs may be useful in wound healing.     

Human corneal fibroblast migration and extracellular matrix synthesis during stromal repair: Role played by platelet‐derived growth factor‐BB,basic fibroblast growth factor,and transforming growth factor‐β1

The development of treatments that modulate corneal wound healing to avoid fibrosis during tissue repair is important for the restoration of corneal transparency after an injury. To date, few studies have studied the influence of growth factors (GFs) on human corneal fibroblast (HCF) expression of extracellular matrix (ECM) proteins such as collagen types I and III, proteoglycans such as perlecan, or proteins implicated in cellular migration such as α5β1‐integrin and syndecan‐4. Using in vitro HCFs, a mechanical wound model was developed to study the influence of the GFs basic fibroblast GF (bFGF), platelet‐derived GF (PDGF‐BB) and transforming GF‐β1 (TGFβ1) on ECM protein production and cellular migration. Our results show that mechanical wounding provokes the autocrine release of bFGF and TGFβ1 at different time points during the wound closure. The HCF response to PDGF‐BB was a rapid closure due to fast cellular migration associated with a high focal adhesion replacement and a high expression of collagen and proteoglycans, producing nonfibrotic healing. bFGF stimulated nonfibrotic ECM production and limited the migration process. Finally, TGFβ1 induced expression of the fibrotic markers collagen type III and α5β1 integrin, and it inhibited cellular migration due to the formation of focal adhesions with a low turnover rate. The novel in vitro HCF mechanical wound model can be used to understand the role played by GFs in human corneal repair. The model can also be used to test the effects of different treatments aimed at improving the healing process. Copyright © 2016 John Wiley & Sons, Ltd.     

Inflammatory response of intervertebral disc cells is reduced by fibrin sealant scaffold in vitro

Intervertebral disc (IVD) degeneration is a complex process characterized by elevated concentrations of proinflammatory cytokines and proteolytic enzymes. Because of pro‐healing constituents, we hypothesized that fibrin sealant (FS) can reduce inflammation and augment soft tissue healing within the damaged or degenerative IVD. To test this, human and porcine nucleus pulposus (NP) and annulus fibrosus (AF) cells were extracted from tissues and encapsulated into alginate beads (NP cells) and type I collagen sponges (AF cells). Half of the alginate and collagen scaffolds were embedded in FS. To provoke inflammatory behaviours, the constructs were cultured with and without continuous IL‐1α (10 ng/ml) for 4, 7 and 14 days. ELISA was used to quantify the cellular synthesis (ng/µg DNA) of clinically relevant cytokines, proteolytic enzymes and growth factors. In NP cell constructs with IL‐1α, the syntheses of TNFα, IL‐1β, IL‐6, IL‐8 was elevated at all culture durations. In the presence of FS, secretion of several pro‐inflammatory cytokines were significantly reduced [IL‐6 and IL‐8 (porcine); and TNFα, IL‐1β, IL‐6, IL‐8 (human)]. Consistent with these reductions, human NP cultures exposed to FS and FS + IL‐1α synthesized significantly reduced amounts of MMP‐1 and ?3 compared to constructs with IL‐1α. For porcine and human AF cells, there were no significant differences in the synthesis of the inflammatory or proteolytic cytokines relative to controls (without IL‐1α) at any culture duration. However, the porcine AF cells exposed to FS synthesized elevated amounts of the anti‐inflammatory cytokine IL‐4. The results suggest that FS may have beneficial effects for patients with degenerated intervertebral discs. Copyright © 2012 John Wiley & Sons, Ltd.     

Platelet‐derived growth factor BB gene‐released scaffolds: biosynthesis and characterization

Tissue engineering generally requires three basic elements; stem/progenitor cells, inductive agents and a biomaterial scaffold; the latter is one of the key components which directly influences cellular activity and matrix formation. Commonly used scaffolds to repair defects in general do not induce stem cell recruitment, which is an essential element to tissue regeneration. In this study, fabrication of a scaffold which is capable of restoring damaged tissue through the recruitment of mesenchymal stem cells (MSCs) by gene therapy of the gene encoding platelet‐derived growth factor‐B (PDGF‐B) was investigated. PDGF‐B adenovirus (AdPDGF) was combined into novel mesoporous bioglass–silk fibrin scaffolds, which were characterized for their controlled release and sustained bioactivity. Our results demonstrate that these scaffolds can release PDGF‐B adenovirus for up to 3 weeks and increase MSC recruitment, both in vitro and following subcutaneous implantation in mice. Osseous calvarial defects in mice further demonstrate the ability of these scaffolds to enhance tissue regeneration through stem cell homing. This study demonstrates the potent ability of host stem cells to regenerate tissue defects through recruitment of MSCs via gene therapy. Copyright © 2013 John Wiley & Sons, Ltd.     

Developing affordable and accessible pro‐angiogenic wound dressings; incorporation of 2 deoxy D‐ribose (2dDR) into cotton fibres and wax‐coated cotton fibres

The absorption capacity of cotton dressings is a critical factor in their widespread use where they help absorb wound exudate. Cotton wax dressings, in contrast, are used for wounds where care is taken to avoid adhesion of dressings to sensitive wounds such as burn injuries. Accordingly, we explored the loading of 2‐deoxy‐D‐ribose (2dDR), a small sugar, which stimulates angiogenesis and wound healing in normal and diabetic rats, into both types of dressings and measured the release of it over several days. The results showed that approximately 90% of 2dDR was released between 3 and 5 days when loaded into cotton dressings. For wax‐coated cotton dressings, several methods of loading of 2dDR were explored. A strategy similar to the commercial wax coating methodology was found the best protocol which provided a sustained release over 5 days. Cytotoxicity analysis of 2dDR loaded cotton dressing showed that the dressing stimulated metabolic activity of fibroblasts over 7 days confirming the non‐toxic nature of this sugar‐loaded dressings. The results of the chick chorioallantoic membrane (CAM) assay demonstrated a strong angiogenic response to both 2dDR loaded cotton dressing and to 2dDR loaded cotton wax dressings. Both dressings were found to increase the number of newly formed blood vessels significantly when observed macroscopically and histologically. We conclude this study offers a simple approach to developing affordable wound dressings as both have the potential to be evaluated as pro‐active dressings to stimulate wound healing in wounds where management of exudate or prevention of adherence to the wounds are clinical requirements.     

Amniotic epithelial cells promote wound healing in mice through high epithelialization and engraftment

Although human amniotic epithelial cells (AMEs) are an attractive source of stem cells, their therapeutic potential in wound healing has not been fully investigated. We evaluated the therapeutic potential of AMEs for wound healing. Real‐time PCR showed that the epithelialization growth factors epidermal growth factor (EGF), platelet‐derived growth factor (PDGF)‐B and chemotactic factors interleukin‐8 (IL‐8 or CXCL8) and neutrophil‐activating protein‐2 (NAP‐2 or CXCL7) were upregulated in AMEs compared with adipose‐derived mesenchymal stem cells (ADMs). In vitro scratch wound assays revealed that AME‐derived conditioned medium substantially accelerated wound closure. Wounds in NOD/SCID mice were created by skin excision, followed by AME transplantation. AMEs implantation significantly accelerated wound healing and increased cellularity and re‐epithelialization. Transplanted AMEs exhibited high engraftment rates and expressed keratinocyte‐specific proteins and cytokeratin in the wound area, suggesting direct benefits for cutaneous closure. Taken together, these data indicate that AMEs possess therapeutic capability for wound healing through the secretion of epithelialization growth factors and enhanced engraftment properties. Copyright © 2015 John Wiley & Sons, Ltd.     

Influence of fibrin matrices and their released factors on epidermal substitute phenotype and engraftment

Cultured epithelial autografts (CEAs) represent a life‐saving surgical technique for full‐thickness skin burns covering more than 60% total body surface area. However, CEAs present numerous drawbacks leading to heavy cosmetic and functional sequelae. In our previous study, we showed that human plasma‐based fibrin matrices (hPBM) could improve the reparative potential of CEAs. Therefore, in the present work, we sought to investigate the role of hPBM compared with fibrin from purified fibrinogen (FPF) or plastic support on epidermal substitute formation and engraftment. The use of hPBM for epidermal substitute culture improved keratinocyte migration, proliferation, and epidermal substitute organization to a better extent than FPF in vitro. Both fibrin matrices favored greater dermal–epidermal junction protein deposition and prevented their degradation. Keratinocyte differentiation was also decreased using both fibrin matrices. Basement membrane protein deposition was mainly influenced by matrix whereas growth factors released from fibrin especially by hPBM were shown to enhance in vitro keratinocyte migration, proliferation, and epidermal substitute organization. Ultimately, epidermal substitutes grown on hPBM displayed better engraftment rates than those cultured on FPF or on plastic support in a NOD‐SCID model of acute wound with the formation of a functional dermal–epidermal junction. Together, these results show the positive impact of fibrin matrices and their released growth factor on epidermal substitute phenotype and grafting efficiency. Fibrin matrices, and especially hPBM, may therefore be of interest to favor the treatment of full‐thickness burn patients.     

Platelet‐rich plasma‐based bioactive membrane as a new advanced wound care tool

Chronic skin ulcers, consequence of diabetes and other pathological conditions, heavily compromise the patient life quality and represent a high and constantly growing cost for National Health Services. Autologous platelet‐rich plasma (PRP), has been proposed to treat these lesions. The absence of guidelines for the PRP production and the need of a fresh preparation for each treatment lead us to develop a protocol for the production of an allogenic PRP‐based bioactive membrane (BAM), standardized for platelet concentration and growth factor release. This work compares BAMs obtained starting from two different platelet concentrations. There was no direct correlation between the amount of growth factors released by BAM in vitro and the initial platelet count. However, different release kinetics were noticed for different growth factors, suggesting that they were differently retained by the two BAMs. The angiogenic potential of both BAMs was determined by Luminex Angiogenesis Assay. The biological activity of the factors released by the two BAMs was confirmed by cell proliferation and migration. A diabetic mouse chronic ulcer model was used to define the best PRP therapeutic dose in vivo. Both BAMs induced wound healing by increasing the thickness of the regenerated epidermis and the vessel number. However, a too high platelet concentration resulted in a slowdown of the membrane resorption that interfered with the skin healing. Overall, the results indicate that the BAMs could represent a natural and effective wound healing tool for the treatment of skin ulcers. Copyright © 2016 John Wiley & Sons, Ltd.     

Repair of an osteochondral defect by sustained delivery of BMP‐2 or TGFβ1 from a bilayered alginate–PLGA scaffold

Regeneration of cartilage defects can be accelerated by localized delivery of appropriate growth factors (GFs) from scaffolds. In the present study we analysed the in vitro and in vivo release rates and delivery efficacies of transforming growth factor‐β1 (TGFβ1) and bone morphogenetic protein‐2 (BMP‐2) from a bilayered system, applied for osteochondral defect repair in a rabbit model. A bone‐orientated, porous PLGA cylinder was overlaid with GF containing PLGA microspheres, dispersed in an alginate matrix. Four microsphere formulations were incorporated: (a) blank ones; (b) microspheres containing 50 ng TGFβ1; (c) microspheres containing 2.5 µg BMP‐2; and (d) microspheres containing 5 µg BMP‐2. Release kinetics and tissue distributions were determined using iodinated (¹²⁵I) GFs. Bioactivity of in vitro released BMP‐2 and TGFβ1 was confirmed in cell‐based assays. In vivo release profiles indicated good GF release control. 20% of BMP‐2 and 15% of TGFβ1 were released during the first day. Virtually the total dose was delivered at the end of week 6. Significant histological differences were observed between untreated and GF‐treated specimens, there being especially relevant short‐term outcomes with 50 ng TGFβ1 and 5 µg BMP‐2. Although the evaluation scores for the newly formed cartilage did not differ significantly, 5 µg BMP‐2 gave rise to higher quality cartilage with improved surface regularity, tissue integration and increased collagen‐type II and aggrecan immunoreactivity 2 weeks post‐implantation. Hence, the bilayered system controlled GF release rates and led to preserved cartilage integrity from 12 weeks up to at least 24 weeks. Copyright © 2012 John Wiley & Sons, Ltd.     

Cytomodulin‐functionalized porous PLGA particulate scaffolds respond better to cell migration,actin production and wound healing in rodent model

In the present study, porous PLGA microparticulate scaffolds (PMS_P), surface‐hydrolysed scaffolds (PMS_Hyd) and cytomodulin‐coupled scaffolds (PMS_CM) were prepared and characterized. After coupling the particles with cytomodulin, the size was reduced from 334 µm (span 0.53) to 278 µm due to hydrolysis, and contact angle also decreased from 70.87 ± 8.56 to 31.43 ± 7.43, indicating an increase in hydrophilicity. Surface roughness and pore density increased, along with an increase in surface area from 9.59 ± 0.36 to 16.82 ± 0.064 m²/g after attaching the biomolecule CM onto the PLGA particles. In vitro cell culture experiments on human dermal fibroblasts (HDFs) were performed for 21 days, in which MTT assay indicated two‐fold higher cell proliferation on PMS_Hyd than on PMS_CM; however, cell distribution, cell spreading and actin production were significantly higher on PMS_CM than on other scaffolds. Migration of cells from PMS_CM to a 2D plate was gradual but the migrated cells attained early confluence, indicating the preservation of normal cellular functions. In a full‐thickness wound mouse model, PMS_CM exhibited 80% wound closure within 2 weeks. Further, at the end of week 3, the inflammatory cell count in the PMS_CM group was reduced to one‐third of the control group, while in PMS_P and PMS_Hyd the extent of inflammation was much higher and more severe. In the case of PMS_CM, abundant fibroblast proliferation, early formation of the scar tissue, eschar formation and inward movement of the wound margins (a zipper‐like movement) towards the deeper layers of the skin suggested advanced wound healing. Cytomodulin‐coupled scaffolds ensured better cell spreading and migration and thus enabled rapid wound healing (see Supporting information, Figure S1). Copyright © 2012 John Wiley & Sons, Ltd.     

Non‐invasive in vivo tracking of fibrin degradation by fluorescence imaging

Fibrin‐based sealants consist of natural coagulation factors involved in the final phase of blood coagulation, during which fibrinogen is enzymatically converted by thrombin to form a solid‐phase fibrin clot. For applications in tissue regeneration, a controlled process of matrix degradation within a certain period of time is essential for optimal wound healing. Hence, it is desirable to follow the kinetics of fibrinolysis at the application site. Non‐invasive molecular imaging systems enable real‐time tracking of processes in the living animal. In this study, a non‐invasive fluorescence based imaging system was applied to follow and quantify site‐specific degradation of fibrin sealant. To enable non‐invasive tracking of fibrin in vivo, fibrin‐matrix was labelled by incorporation of a fluorophore‐conjugated fibrinogen component. Protein degradation and release of fluorescence were, in a first step, correlated in vitro. In vivo, fluorophore‐labelled fibrin was subcutaneously implanted in mice and followed throughout the experiment using a multispectral imaging system. For the fluorescent fibrin, degradation correlated with the release of fluorescence from the clots in vitro. In vivo it was possible to follow and quantify implanted fibrin clots throughout the experiment, demonstrating degradation kinetics of approximately 16 days in the subcutaneous compartment, which was further confirmed by histological evaluation of the application site. Copyright © 2014 John Wiley & Sons, Ltd.     

Tissue‐regenerating functions of coagulation factor XIII

The protransglutaminase factor XIII (FXIII) has recently attracted attention within the field of tissue regeneration, as it has been found that FXIII significantly influences wound healing by exerting a multitude of functions. It supports hemostasis by enhancing platelet adhesion to damaged endothelium, and by its cross‐linking activity it stabilizes the formed fibrin clot. Furthermore, FXIII limits bacterial dissemination from the wound and incorporates macromolecules of importance for cellular infiltration, supporting cell migration and survival. FXIII‐mediated complex formation of the vascular endothelial growth factor receptor 2 and α_Vβ₃ integrin is important for angiogenesis, supporting the formation of granulation tissue. Chronic inflammatory conditions involving bleeding and activation of the coagulation cascade have been shown to lead to reduced FXIII levels in plasma. Of particular importance for this review is the fact that patients suffering from inflammatory bowel disease (IBD) have reduced FXIII antigen levels and activity. Furthermore, these patients show impaired mucosal healing, which supports the inflammatory state of the disease. This review summarizes the role of FXIII in the healing of wounds, and briefly summarizes the previous use of FXIII in clinical settings. Moreover, it addresses the potential role for FXIII as a therapeutic agent in the healing of persistent wounds during chronic conditions, with an emphasis on IBD.     

Towards embryonic‐like scaffolds for skin tissue engineering: identification of effector molecules and construction of scaffolds

Autologous skin grafts are the gold standard for the treatment of burn wounds. In a number of cases, treatment with autologous tissue is not possible and skin substitutes are used. The outcome, however, is not optimal and improvements are needed. Inspired by scarless healing in early embryonic development, we here set out a strategy for the design and construction of embryonic‐like scaffolds for skin tissue engineering. This strategy may serve as a general approach in the construction of embryonic‐like scaffolds for other tissues/organ. As a first step, key effector molecules upregulated during embryonic and neonatal skin formation were identified using a comparative gene expressing analysis. A set of 20 effector molecules was identified, from which insulin‐like growth factor 2 (IGF2) and sonic hedgehog (SHH) were selected for incorporation into a type I collagen–heparin scaffold. Porous scaffolds were constructed using purified collagen fibrils and 6% covalently bound heparin (to bind and protect the growth factors), and IGF2 and SHH were incorporated either individually (~0.7 and 0.4 µg/mg scaffolds) or in combination (combined ~1.5 µg/mg scaffolds). In addition, scaffolds containing hyaluronan (up to 20 µg/mg scaffold) were prepared, based on the up‐ or downregulation of genes involved in hyaluronan synthesis/degradation and its suggested role in scarless healing. In conclusion, based on a comprehensive gene expression analysis, a set of effector molecules and matrix molecules was identified and incorporated into porous scaffolds. The scaffolds thus prepared may create an 'embryonic‐like' environment for cells to recapitulate embryonic events and for new tissues/organs. Copyright © 2013 John Wiley & Sons, Ltd.     

Conditioned media from hypoxic‐cultured human dental pulp cells promotes bone healing during distraction osteogenesis

Distraction osteogenesis (DO) is a surgical procedure used to correct various skeletal disorders. Improving the technique by reducing the healing time would be of clinical relevance. The aim of this study was to determine the angiogenic and regenerative potential of conditioned media (CMs) collected from human dental pulp cells (hDPCs) grown under different culture conditions. CM collected from cells under hypoxia was used to improve bone healing and the DO procedure in vivo . The angiogenic potentials of CMs collected from hDPCs grown under normoxic (?Nor) and hypoxic (?Hyp) conditions were evaluated by quantitative PCR (VEGF‐A , angiopoietin‐1, angiopoietin‐2, interleukin‐6 (IL‐6 ) and CXCL12 ), ELISA assays (VEGF‐A, Ang‐2), tube‐formation and wound‐healing assays, using human umbilical vein endothelial cells. The results demonstrated that hypoxic CM had significantly higher angiogenic potential than normoxic CM. Human fetal osteoblasts (hFOBs) were exposed to CM, followed by alizarin red staining, to assess the osteogenic potential. It was found that CM did not enhance the mineralization capacity of hFOBs. DO was performed in the tibiae of 30 mice, followed by a local injection of 20 µl CM (CM–Nor and CM–Hyp groups) or serum‐free DMEM (control group) into the distraction zone every second day. The mice were sacrificed at days 13 and 27. The CM–Hyp treatment revealed a higher X‐ray density than the control group (p < 0.05). Our study suggests that the angiogenic effect promoted by hypoxic culture conditions is dependent on VEGF‐A and Ang‐2 released from hDPCs. Furthermore, CM–Hyp treatment may thus improve the DO procedure, accelerating bone healing. © 2015 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons, Ltd.