Microfluidic evidence of synergistic effects between mesenchymal stromal cell-derived biochemical factors and biomechanical forces to control endothelial cell function.

A functional vascular system is a prerequisite for bone repair as disturbed angiogenesis often causes non-union. Paracrine factors released from human bone marrow derived mesenchymal stromal cells (BMSCs) have angiogenic effects on endothelial cells. However, whether these paracrine factors participate in blood flow dynamics within bone capillaries remains poorly understood. Here, we used two different microfluidic designs to investigate critical steps during angiogenesis and found pronounced effects of endothelial cell proliferation as well as chemotactic and mechanotactic migration induced by BMSC conditioned medium (CM). The application of BMSC-CM in dynamic cultures demonstrates that bioactive factors in combination with fluidic flow-induced biomechanical signals significantly enhanced endothelial cell migration. Transcriptional analyses of endothelial cells demonstrate the induction of a unique gene expression profile related to tricarboxylic acid cycle and energy metabolism by the combination of BMSC-CM factors and shear stress, which opens an interesting avenue to explore during fracture healing. Our results stress the importance of in vivo - like microenvironments simultaneously including biochemical, biomechanical and oxygen levels when investigating key events during vessel repair. STATEMENT OF SIGNIFICANCE: Our results demonstrate the importance of recapitulating in vivo - like microenvironments when investigating key events during vessel repair. Endothelial cells exhibit enhanced angiogenesis characteristics when simultaneous exposing them to hMSC-CM, mechanical forces and biochemical signals simultaneously. The improved angiogenesis may not only result from the direct effect of growth factors, but also by reprogramming of endothelial cell metabolism. Moreover, with this model we demonstrated a synergistic impact of mechanical forces and biochemical factors on endothelial cell behavior and the expression of genes involved in the TCA cycle and energy metabolism, which opens an interesting new avenue to stimulate angiogenesis during fracture healing.

Heparan sulfate glycosaminoglycan mimetic improves pressure ulcer healing in a rat model of cutaneous ischemia-reperfusion injury.

Pressure ulcers are a major clinical problem, with a large burden on healthcare resources. This study evaluated the effects of the heparan sulfate glycosaminoglycan mimetic, OTR4120, on pressure ulceration and healing. Ischemia-reperfusion (I-R) was evoked to induce pressure ulcers by external clamping and then removal of a pair of magnet disks on rat dorsal skin for a single ischemic period of 16 hours. Immediately after magnet removal, rats received an intramuscular injection of OTR4120 weekly for up to 1 month. During the ischemic period, normal skin perfusion was reduced by at least 60% and at least 20-45% reperfused into the ischemic region after compression release. This model caused sustained skin incomplete necrosis for up to 14 days and led to grade 2-3 ulcers. OTR4120 treatment decreased the area of skin incomplete necrosis and degree of ulceration. OTR4120 treatment also reduced inflammation and increased angiogenesis. In OTR4120-treated ulcers, the contents of vascular endothelial growth factor, platelet-derived growth factor, and transforming growth factor beta-1 were increased. Moreover, OTR4120 treatment promoted early expression of alpha-smooth muscle actin and increased collagen biosynthesis. Long-term restoration of wounded tissue biomechanical strength was significantly enhanced after OTR4120 treatment. Taken together, we conclude that OTR4120 treatment reduces pressure ulcer formation and potentiates the internal healing bioavailability.

Stimulated neovascularization, inflammation resolution and collagen maturation in healing rat cutaneous wounds by a heparan sulfate glycosaminoglycan mimetic, OTR4120.

Heparan sulfate glycosaminoglycans (HS-GAGs) are not only the structural elements of tissue architecture but also regulate the bioavailability and transduction pathways of heparan sulfate-bound polypeptides released by cells or the extracellular matrix. Heparan sulfate-bound polypeptides include inflammatory mediators, chemokines, angiogenic factors, morphogens, and growth-promoting factors that induce cell migration, proliferation, and differentiation in wound healing. OTR4120, a polymer engineered to mimic the properties of HS-GAGs, is used to replace the natural HS-GAGs that are degraded during wound repair, and enhance the tissue regeneration by preserving the cellular microenvironment and the endogenous signals needed for tissue regeneration. We previously demonstrated that OTR4120 treatment had a long-term effect on increasing breaking strength and vasodilation in healing rat full-thickness excisional wounds. The present study investigates the underlying mechanisms of the effects of OTR4120 treatment in improving the quality of cutaneous wound repair. We found that OTR4120 treatment stimulated inflammation resolution and increased neovascularization. OTR4120 treatment also promoted epidermal migration and proliferation during reepithelialization. Moreover, the granulation tissue formation and collagen maturation were improved in OTR4120-treated wounds. Three months after wounding, the effects of OTR4120 treatment on vascularization and inflammation resolution were normalized, except for an improved neodermis. We conclude that OTR4120 is a potential matrix therapeutic agent that ensures the quality of normal cutaneous wound repair and may restore impaired wound healing characterized by deficient angiogenesis and prolonged inflammation.

Novel experimental surgical strategy to prevent traumatic neuroma formation by combining a 3D-printed Y-tube with an autograft.

OBJECTIVE Traumatic neuromas may develop after nerve injury at the proximal nerve stump, which can lead to neuropathic pain. These neuromas are often resistant to therapy, and excision of the neuroma frequently leads to recurrence. In this study, the authors present a novel surgical strategy to prevent neuroma formation based on the principle of centro-central anastomosis (CCA), but rather than directly connecting the nerve ends to an autograft, they created a loop using a 3D-printed polyethylene Y-shaped conduit with an autograft in the distal outlets. METHODS The 3D-printed Y-tube with autograft was investigated in a model of rat sciatic nerve transection in which the Y-tube was placed on the proximal sciatic nerve stump and a peroneal graft was placed between the distal outlets of the Y-tube to form a closed loop. This model was compared with a CCA model, in which a loop was created between the proximal tibial and peroneal nerves with a peroneal autograft. Additional control groups consisted of the closed Y-tube and the extended-arm Y-tube. Results were analyzed at 12 weeks of survival using nerve morphometry for the occurrence of neuroma formation and axonal regeneration in plastic semi-thin sections. RESULTS Among the different surgical groups, the Y-tube with interposed autograft was the only model that did not result in neuroma formation at 12 weeks of survival. In addition, a 13% reduction in the number of myelinated axons regenerating through the interposed autograft was observed in the Y-tube with autograft model. In the CCA model, the authors also observed a decrease of 17% in the number of myelinated axons, but neuroma formation was present in this model. The closed Y-tube resulted in minimal nerve regeneration inside the tube together with extensive neuroma formation before the entrance of the tube. The extended-arm Y-tube model clearly showed that the majority of the regenerating axons merged into the Y-tube arm, which was connected to the autograft, leaving the extended plastic arm almost empty. CONCLUSIONS This pilot study shows that our novel 3D-printed Y-tube model with interposed autograft prevents neuroma formation, making this a promising surgical tool for the management of traumatic neuromas.

Hyperbaric oxygen therapy for wound healing in diabetic rats: Varying efficacy after a clinically-based protocol.

Hyperbaric oxygen therapy (HBOT) is a clinical treatment in which a patient breathes pure oxygen for a limited period of time at an increased pressure. Although this therapy has been used for decades to assist wound healing, its efficacy for many conditions is unproven and its mechanism of action is not yet fully clarified. This study investigated the effects of HBOT on wound healing using a diabetes-impaired pressure ulcer rat model. Seven weeks after streptozotocin-induced diabetes in rats (n = 55), a pressure ulcer was created on dorsal skin. Subsequently, animals received HBOT during 6 weeks following a standard clinical protocol (HBOT group with varying endpoints up to 42 days post-wounding) versus controls without HBOT. Capillary venous oxygen saturation (SO2) showed a significant increase in the HBOT group on day 24; however, this increase was significant at this time point only. The quantity of hemoglobin in the micro-blood vessels (rHB) showed a significant decrease in the HBOT group on days 21 and 42, and showed a trend to decrease on day 31. Blood flow in the microcirculation showed a significant increase on days 17, 21 and 31 but a significant decrease on days 24 and 28. Inflammation scoring showed significantly decreased CD68 counts in the HBOT group on day 42, but not in the early stages of wound healing. Animals in the HBOT group showed a trend for an increase in mean wound breaking strength on day 42.

Fibrin improves skin wound perfusion in a diabetic rat model.

The fibrin matrix of the thrombus that is formed directly after wounding, is an important determinant of the success of the early phase of wound healing. This phase is often impaired in patients with diabetes. A promising approach to improve skin wound healing is the application of a pro-angiogenic fibrin matrix onto the wound. We studied this in 59 female WAG/RijCrl diabetic rats, in which we created two dorsal full-thickness wounds of which one was treated with a human physiological fibrin matrix (2mg/ml) and one with PBS as control. Wound healing parameters were determined at different time points. The wound closure was significantly improved in fibrin-treated wounds on day 3 and 7. Also, fibrin-treated wounds showed a significantly higher perfusion on day 28 and 35 compared to control wounds (p<0.05). CD68 staining revealed that human fibrin did not induce an immune response. IN CONCLUSION: the application of a fibrin matrix on a diabetic wound showed improved perfusion and an increased early closure rate of the wound area.

Studying skin tumourigenesis and progression in immunocompetent hairless SKH1-hr mice using chronic 7,12-dimethylbenz(a)anthracene topical applications to develop a useful experimental skin cancer model.

Previous studies have established that 7,12-dimethylbenz(a)anthracene (DMBA) can initiate skin tumourigenesis in conventional furred mouse models by acting on hair follicle stem cells. However, further cancer progression depends on repeated applications of tumour promoter agents. This study evaluated the timeline involved in skin tumourigenesis and progression in immunocompetent hairless SKH1-hr mice with dysfunctional hair follicles using only DMBA with no additional tumour promoter agents. The results showed that topical application of 30 µg (117 nmol) of DMBA over the back and flank regions of the mouse once a week and 15 µg (58.5 nmol) twice a week produced skin tumours after 7-8 weeks. However, by week 14 a heavy benign tumour load required the mice to be euthanized. Lowering the DMBA dose to 15 µg (58.5 nmol) once a week produced tumours more slowly and allowed the mice to be studied for a longer period to week 23. This low-dose DMBA regimen yielded a high percentage of malignant tumours (58.8%) after 23 weekly applications. Additionally DMBA-treated skin showed an increase in mean epidermal thickness in comparison to untreated and acetone-treated skin. Despite the aberrant hair follicles in SKH1-hr mice, this chemically driven skin cancer model in hairless mice can serve as a suitable alternative to the ultraviolet-induced skin cancer models and can be reliably replicated as demonstrated by both the pilot and main experiments.

Specific effects of fibrinogen and the γA and γ'-chain fibrinogen variants on angiogenesis and wound healing.

In a newly formed wound, the natural fibrin network provides the first temporary matrix for tissue repair. Topical application of fibrin to a new wound may improve wound healing. A matrix of the common natural γ' fibrin variant may further improve wound healing because it is expected to have a different architecture and this will influence angiogenesis, because it possesses increased thrombin and factor XIII binding and decreased platelet binding, when compared with the common γA fibrin matrix. Our objective was to determine the effect of fibrinogen and its γA and γ' variants on angiogenesis and wound healing. We used in vitro angiogenesis models and an in vivo rat full-thickness excisional wound healing model. When comparing γA and γ' fibrin in vitro, more tube-like structures were formed on day 7 in γA fibrin than in γ' fibrin (13.83±6.12 AU vs. 6.1±1.46 AU). Wounds treated with fibrin demonstrated improved healing in vivo with more perfusion (47%±3% vs. 26%±4%, p<0.01 in placebo) and higher CD34 density score (2.0±0.4 vs. 2.8±0.1, p<0.01) on day 21 with fibrin matrices when compared with placebo-treated wounds. Increased perfusion was observed in γA fibrin-treated wounds on day 21 (53%±10% vs. 41%±7% for γ' fibrin). The other parameters showed slightly improved (not significant) wound healing with γA fibrin compared with γ' fibrin matrices. In conclusion, the use of fibrin and fibrin variant matrices offers an interesting methodology to stimulate the wound healing process.

Hyperbaric oxygen therapy to treat diabetes impaired wound healing in rats.

Wound healing in diabetes is frequently impaired and its treatment remains a challenge. Hyperbaric oxygen therapy (HBOT) receives a wide attendance and is often used as a last resort treatment option, however, its effectiveness for many conditions is unproven. We tested the effect of HBOT on healing of diabetic ulcers in an animal experimental setting. Experimental diabetes was induced by intraperitoneal injection of streptozotocin. Four weeks after diabetes induction, rats were ulcerated by clamping a pair of magnet disks on the dorsal skin for 16 h. After magnet removal, the animals received HBOT, daily on weekdays, for 4 weeks. To examine the effect of HBOT on diabetes impaired wound healing, the degree of wound tissue perfusion, inflammation, angiogenesis, and tissue breaking strength were evaluated. HBOT effects on the degree of inflammation and number of blood vessels could not be observed. HBOT improved the tissue breaking strength of the wound, however, this did not reach statistical significance. Twenty hours after ending the HBOT, a significantly improved oxygen saturation of the hemoglobin at the venous end of the capillaries and the quantity of hemoglobin in the micro-blood vessels was measured.

Hyperbaric oxygen treatment of tissue-engineered mucosa enhances secretion of angiogenic factors in vitro.

The survival of tissue-engineered mucosa (TEM) after implantation is mostly dependent on the presence of blood vessels for continuous oxygen supply. Therefore the stimulation of vascularization of TEM is essential to improve survival in vivo. Hyperbaric oxygen (HBO) treatment, used to improve wound healing, stimulates the secretion of angiogenic factors. In this study we evaluated the effect of daily HBO treatments on TEM for 1, 3, or 5 consecutive days. Overall histology with hematoxylin-eosin staining showed no apparent changes after one treatment. After three and five HBO treatments, the basal layer became irregular and pyknotic cells were observed. Measurements of the viable epithelium showed significant thinning after one and five treatments, however, proliferation was not affected. The angiogenic factors keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), basic fibroblast growth factor (FGFbasic), and placental growth factor (PlGF) were significantly increased after one HBO treatment, whereas after three treatments a significant decrease of FGFbasic and PlGF was seen. After five treatments KGF, PlGF, and vascular endothelial growth factor (VEGF) were significantly increased. One HBO treatment of TEM enhances the secretion of important angiogenic factors, hereby potentially improving the survival rate after in vivo implantation.

Diabetes-impaired wound healing is improved by matrix therapy with heparan sulfate glycosaminoglycan mimetic OTR4120 in rats.

Wound healing in diabetes is frequently impaired, and its treatment remains a challenge. We tested a therapeutic strategy of potentiating intrinsic tissue regeneration by restoring the wound cellular environment using a heparan sulfate glycosaminoglycan mimetic, OTR4120. The effect of OTR4120 on healing of diabetic ulcers was investigated. Experimental diabetes was induced by intraperitoneal injection of streptozotocin. Seven weeks after induction of diabetes, rats were ulcerated by clamping a pair of magnet disks on the dorsal skin for 16 h. After magnet removal, OTR4120 was administered via an intramuscular injection weekly for up to 4 weeks. To examine the effect of OTR4120 treatment on wound heal-ing, the degree of ulceration, inflammation, angiogenesis, and collagen synthesis were evaluated. We found that OTR4120 treatment significantly reduced the degree of ulceration and the time of healing. These effects were associated with reduced neutrophil infiltration and macrophage accumulation and enhanced angiogenesis. OTR4120 treatment also increased the collagen content with an increase of collagen type I biosynthesis and reduction of collagen type III biosynthesis. Moreover, restoration of the ulcer biomechanical strength was significantly enhanced after OTR4120 treatment. This study shows that matrix therapy with OTR4120 improves diabetes-impaired wound healing.