Development of ELIP to Assess Physical Literacy for Emerging Adults: A Methodological and Epistemological Challenge.

Purpose: Following increased interest in physical literacy (PL), development of appropriate tools for assessment has become an important next step for its operationalization. To forward the development of such tools, the objective of this study was to build the foundations of the Évaluation de la Littératie Physique (ELIP), designed to help reduce existing tensions in approaches to PL assessment that may be resulting in a low uptake into applied settings. Methods: We followed two steps: (1) the development of the first version of ELIP by deploying a Delphi method (n = 30); and (2) the modification of items through cognitive interviews with emerging adults (n = 32). Results: The expert consensus highlighted four dimensions of PL to be assessed-physical; affective; cognitive; and social-with new perspectives, including a preference for broad motor tests over fitness. Conclusion: Results offer new insights into the assessment of emerging adults' PL, but ELIP still requires further work concerning validity, reliability, and sensitivity.

Amlodipine accelerates bone healing in a stable closed femoral fracture model in mice.

Calcium channel blockers (CCBs), which are widely used in the treatment of hypertension, have been shown to influence bone metabolism. However, there is little information on whether CCBs also influence the process of fracture healing. Therefore, the effect of the CCB amlodipine on bone healing was studied in a stable closed fracture model in mice using intramedullary screw fixation. Bone healing was investigated by radiology, biomechanics, histomorphometry and Western blot analysis 2 and 5 weeks after fracture healing. Animals were treated daily (post operatively) per os using a gavage with amlodipine low dose (1 mg/ kg body weight, n = 20), amlodipine high dose (3 mg/kg body weight, n = 20) or vehicle (NaCl) (control, n = 20) serving as a negative control. At 2 and 5 weeks, histomorphometric analysis revealed a significantly larger amount of bone tissue within the callus of amlodipine low-dose- and high-dose-treated animals when compared to controls. This was associated with a smaller amount of cartilaginous and fibrous tissue, indicating an acceleration of fracture healing. Biomechanics showed a slightly, but not significantly, higher bending stiffness in amlodipine low-dose- and high-dose-treated animals. Western blot analysis revealed a significantly increased expression of bone morphogenetic protein (BMP)-2 and vascular endothelial growth factor (VEGF). Moreover, the analysis showed a 5-fold higher expression of osteoprotegerin (OPG) and a 10-fold elevated expression of the receptor activator of NF-κB ligand (RANKL), indicating an increased bone turnover. These findings demonstrated that amlodipine accelerated fracture healing by stimulating bone formation, callus remodelling and osteoclast activity.

Local Application of Mineral-Coated Microparticles Loaded With VEGF and BMP-2 Induces the Healing of Murine Atrophic Non-Unions.

Deficient angiogenesis and disturbed osteogenesis are key factors for the development of nonunions. Mineral-coated microparticles (MCM) represent a sophisticated carrier system for the delivery of vascular endothelial growth factor (VEGF) and bone morphogenetic protein (BMP)-2. In this study, we investigated whether a combination of VEGF- and BMP-2-loaded MCM (MCM + VB) with a ratio of 1:2 improves bone repair in non-unions. For this purpose, we applied MCM + VB or unloaded MCM in a murine non-union model and studied the process of bone healing by means of radiological, biomechanical, histomorphometric, immunohistochemical and Western blot techniques after 14 and 70 days. MCM-free non-unions served as controls. Bone defects treated with MCM + VB exhibited osseous bridging, an improved biomechanical stiffness, an increased bone volume within the callus including ongoing mineralization, increased vascularization, and a histologically larger total periosteal callus area consisting predominantly of osseous tissue when compared to defects of the other groups. Western blot analyses on day 14 revealed a higher expression of osteoprotegerin (OPG) and vice versa reduced expression of receptor activator of NF-κB ligand (RANKL) in bone defects treated with MCM + VB. On day 70, these defects exhibited an increased expression of erythropoietin (EPO), EPO-receptor and BMP-4. These findings indicate that the use of MCM for spatiotemporal controlled delivery of VEGF and BMP-2 shows great potential to improve bone healing in atrophic non-unions by promoting angiogenesis and osteogenesis as well as reducing early osteoclast activity.

High glucose exposure promotes proliferation and in vivo network formation of adipose-tissue-derived microvascular fragments.

High glucose concentrations have been shown to activate endothelial cells and promote angiogenesis. In the present study, it was investigated whether high glucose concentrations could improve the vascularisation capacity of adipose-tissue-derived microvascular fragments (ad-MVF). Ad-MVF were isolated from the epididymal fat pads of donor mice and cultivated for 24 h in University of Wisconsin (UW) solution supplemented with vehicle or 30 mM glucose. Protein expression, morphology, viability and proliferation of the cultivated ad-MVF were analysed by means of proteome profiler mouse angiogenesis array, scanning electron microscopy and immunohistochemistry. Additional cultivated ad-MVF were seeded on to collagen-glycosaminoglycan scaffolds to study their in vivo vascularisation capacity in the dorsal skinfold chamber model by intravital fluorescence microscopy, histology and immunohistochemistry. In vitro, high glucose exposure changed the protein expression pattern of ad-MVF with endoglin, interleukin (IL)-1ß and monocyte chemoattractant protein (MCP)-1 as the most up-regulated pro-angiogenic factors. Moreover, high glucose exposure induced the formation of nanopores in the ad-MVF wall. In addition, ad-MVF contained significantly larger numbers of proliferating endothelial and perivascular cells while exhibiting a comparable number of apoptotic cells when compared to vehicle-treated controls. In vivo, scaffolds seeded with high-glucose-exposed ad-MVF exhibited an improved vascularisation and tissue incorporation. These findings demonstrated that the exposure of cultivated ad-MVF to high glucose concentrations is a promising approach to improve their in vivo performance as vascularisation units for tissue engineering and regenerative medicine.

Erythropoietin does not improve fracture healing in aged mice.

Fracture healing in the elderly is associated with a declined healing potential caused by multiple factors including a delay of vascularization. Erythropoietin (EPO) has been demonstrated to improve vascularization and fracture healing in adult mice. We, therefore, hypothesized that EPO in aged mice also improves fracture healing. For this purpose, EPO was given daily in a femoral fracture model in aged mice and compared to vehicle-treated controls using radiological, biomechanical, histomorphometric and Western blot techniques. Blood analyses revealed significantly higher concentrations of hemoglobin and a higher hematocrit in EPO-treated animals at 14 and 35 days after fracture. Micro-computed tomography (µCT) indicated that the fraction of bone volume/tissue volume within the callus did not differ between the two groups. However, µCT showed a 3-fold increased tissue mineral density (TMD) in the callus of EPO-treated animals compared to controls. The callus TMD of the EPO-treated animals was also 2-fold higher when compared to the TMD of the unfractured contralateral femur. Interestingly, biomechanical analyses revealed a reduced bending stiffness in femurs of EPO-treated animals at day 35. The histomorphometrically analyzed callus size and callus composition did not show significant differences between the study groups. However, Western blot analyses exhibited an increased expression of osteoprotegerin (OPG), but in particular of receptor activator of NF-κB ligand (RANKL) in the callus of the EPO-treated animals. Further histological analyses of the callus tissue showed that this was associated with an increased number of newly formed blood vessels and a higher number of tartrate-resistant acid phosphatase (TRAP)+ cells. Conclusion: In fracture healing of aged mice EPO treatment increases callus TMD as well as OPG and RANKL expression, indicating an accelerated bone turnover when compared to controls. However, EPO does not improve fracture healing in aged mice. The process of fracture healing may be altered by EPO due to a deterioration of the microcirculation caused by the worsened rheological properties of the blood and due to an increased bone fragility caused by the accelerated bone turnover. Thus, EPO may not be used to improve fracture healing in the elderly.

Effects of locally applied adipose tissue-derived microvascular fragments by thermoresponsive hydrogel on bone healing.

Insufficient vascularization is a major cause for the development of non-unions. To overcome this problem, adipose tissue-derived microvascular fragments (MVF) may serve as vascularization units. However, their application into bone defects needs a carrier system. Herein, we analyzed whether this is achieved by a thermoresponsive hydrogel (TRH). MVF were isolated from CD-1 mice and cultivated after incorporation into TRH, while non-incorporated MVF served as controls. Viability of MVF was assessed immunohistochemically over a 7-day period. Moreover, osteotomies were induced in femurs of CD-1 mice. The osteotomy gaps were filled with MVF-loaded TRH (TRH + MVF), unloaded TRH (TRH) or no material (control). Bone healing was evaluated 14 and 35 days postoperatively. MVF incorporated into TRH exhibited less apoptotic cells and showed a stable vessel morphology compared to controls. Micro-computed tomography revealed a reduced bone volume in TRH + MVF femurs. Histomorphometry showed less bone and more fibrous tissue after 35 days in TRH + MVF femurs compared to controls. Accordingly, TRH + MVF femurs exhibited a lower osseous bridging score and a reduced bending stiffness. Histology and Western blot analysis revealed an increased vascularization and CD31 expression, whereas vascular endothelial growth factor (VEGF) expression was reduced in TRH + MVF femurs. Furthermore, the callus of TRH + MVF femurs showed increased receptor activator of NF-κB ligand expression and higher numbers of osteoclasts. These findings indicate that TRH is an appropriate carrier system for MVF. Application of TRH + MVF increases the vascularization of bone defects. However, this impairs bone healing, most likely due to lower VEGF expression during the early course of bone healing. STATEMENT OF SIGNIFICANCE: In the present study we analyzed for the first time the in vivo performance of a thermoresponsive hydrogel (TRH) as a delivery system for bioactive microvascular fragments (MVF). We found that TRH represents an appropriate carrier for MVF as vascularization units and maintains their viability. Application of MVF-loaded TRH impaired bone formation in an established murine model of bone healing, although vascularization was improved. This unexpected outcome was most likely due to a reduced VEGF expression in the early phase bone healing.

Erythropoietin promotes network formation of transplanted adipose tissue-derived microvascular fragments.

The seeding of tissue constructs with adipose tissue-derived microvascular fragments (ad-MVF) is an emerging pre-vascularisation strategy. Ad-MVF rapidly reassemble into new microvascular networks after in vivo implantation. Herein it was analysed whether this process was improved by erythropoietin (EPO). Ad-MVF were isolated from green fluorescent protein (GFP)+ as well as wild-type C57BL/6 mice and cultivated for 24 h in medium supplemented with EPO (20 IU/mL) or vehicle. Freshly isolated, non-cultivated ad-MVF served as controls. Protein expression, cell viability and proliferation of ad-MVF were assessed by proteome profiler array and fluorescence microscopy. GFP+ ad-MVF were seeded on collagen-glycosaminoglycan matrices, which were implanted into dorsal skinfold chambers of C57BL/6 mice, to analyse their vascularisation over 14 d by intravital fluorescence microscopy, histology and immunohistochemistry. Cultivation up-regulated the expression of pro- and anti-angiogenic factors within both vehicle- and EPO-treated ad-MVF when compared with non-cultivated controls. Moreover, EPO treatment suppressed cultivation-associated apoptosis and significantly increased the number of proliferating endothelial cells in ad-MVF when compared with vehicle-treated and non-cultivated ad-MVF. Accordingly, implanted matrices seeded with EPO-treated ad-MVF exhibited an improved vascularisation, as indicated by a significantly higher functional microvessel density. The matrices of the three groups contained a comparably large fraction of GFP+ microvessels originating from the ad-MVF, whereas the tissue surrounding the matrices seeded with EPO-treated ad-MVF exhibited a significantly increased microvessel density when compared with the other two groups. These findings indicated that EPO represents a promising cytokine to further boost the excellent vascularisation properties of ad-MVF in tissue-engineering applications.

High-intensity interval training improves insulin sensitivity in older individuals.

AIM: Metabolic health may deteriorate with age as a result of altered body composition and decreased physical activity. Endurance exercise is known to counter these changes delaying or even preventing onset of metabolic diseases. High-intensity interval training (HIIT) is a time efficient alternative to regular endurance exercise, and the aim of this study was to investigate the metabolic benefit of HIIT in older subjects. METHODS: Twenty-two sedentary male (n = 11) and female (n = 11) subjects aged 63 ± 1 years performed HIIT training three times/week for 6 weeks on a bicycle ergometer. Each HIIT session consisted of five 1-minute intervals interspersed with 1½-minute rest. Prior to the first and after the last HIIT session whole-body insulin sensitivity, measured by a hyperinsulinaemic-euglycaemic clamp, plasma lipid levels, HbA1c, glycaemic parameters, body composition and maximal oxygen uptake were assessed. Muscle biopsies were obtained wherefrom content of glycogen and proteins involved in muscle glucose handling were determined. RESULTS: Insulin sensitivity (P = .011) and maximal oxygen uptake increased (P < .05) in both genders, while plasma cholesterol (P < .05), low-density lipoprotein (P < .05), visceral fat mass (P < .05) and per cent body fat (P < .05) decreased after 6 weeks of HIIT. HbA1c decreased only in males (P = .001). Muscle glycogen content increased in both genders (P = .001) and in line GLUT4 (P < .05), glycogen synthase (P = .001) and hexokinase II (P < .05) content all increased. CONCLUSION: Six weeks of HIIT significantly improves metabolic health in older males and females by reducing age-related risk factors for cardiometabolic disease.

BMP-2-coated mineral coated microparticles improve bone repair in atrophic non-unions.

Atrophic non-unions are a major clinical problem. Mineral coated microparticles (MCM) are electrolyte-coated hydroxyapatite particles that have been shown in vitro to bind growth factors electrostatically and enable a tuneable sustained release. Herein, we studied whether MCM can be used in vivo to apply Bone Morphogenetic Protein-2 (BMP-2) to improve bone repair of atrophic non-unions. For this purpose, atrophic non-unions were induced in femurs of CD-1 mice (n = 48). Animals either received BMP-2-coated MCM (MCM + BMP; n = 16), uncoated MCM (MCM; n = 16) or no MCM (NONE; n = 16). Bone healing was evaluated 2 and 10 weeks postoperatively by micro-computed tomographic (µCT), biomechanical, histomorphometric and immunohistochemical analyses. µCT revealed more bone volume with more highly mineralised bone in MCM + BMP femurs. Femurs of MCM + BMP animals showed a significantly higher bending stiffness compared to other groups. Histomorphometry further demonstrated that the callus of MCM + BMP femurs was larger and contained more bone and less fibrous tissue. After 10 weeks, 7 of 8 MCM + BMP femurs presented with complete osseous bridging, whereas NONE femurs exhibited a non-union rate of 100 %. Of interest, immunohistochemistry could not detect macrophages within the callus, indicating a good biocompatibility of MCM. In conclusion, the local application of BMP-2-coated MCM improved bone healing in a challenging murine non-union model and, thus, should be of clinical interest in the treatment of non-unions.

Effects of macrophage-activating lipopeptide-2 (MALP-2) on the vascularisation of implanted polyurethane scaffolds seeded with microvascular fragments.

The seeding of scaffolds with adipose tissue-derived microvascular fragments represents a promising strategy to establish a sufficient blood supply in tissue constructs. Herein, we analysed whether a single application of macrophage-activating lipopeptide-2 (MALP-2) at the implantation site further improves the early vascularisation of such microvessel-seeded constructs. Microvascular fragments were isolated from epididymal fat pads of C57BL/6 mice. The fragments were seeded on polyurethane scaffolds, which were implanted into mouse dorsal skinfold chambers exposed to MALP-2 or vehicle (control). The inflammatory host tissue response and the vascularisation of the scaffolds were analysed using intravital fluorescence microscopy, histology and immunohistochemistry. We found that the numbers of microvascular adherent leukocytes were significantly increased in MALP-2-treated chambers during the first 3 days after scaffold implantation when compared to controls. This temporary inflammation resulted in an improved vascularisation of the host tissue surrounding the implants, as indicated by a higher density of CD31-positive microvessels at day 14. However, the MALP-2-exposed scaffolds themselves presented with a lower functional microvessel density in their centre. In addition, in vitro analyses revealed that MALP-2 promotes apoptotic cell death of endothelial and perivascular cells in isolated microvascular fragments. Hence, despite the beneficial pro-angiogenic properties of MALP-2 at the implantation site, the herein evaluated approach may not be recommended to improve the vascularisation capacity of microvascular fragments in tissue engineering applications.

Obesity does not affect the healing of femur fractures in mice.

Obesity is reported to be both protective and deleterious to bone. Lipotoxicity and inflammation might be responsible for bone loss through inhibition of osteoblasts and activation of osteoclasts. However, little is known whether obesity affects the process of fracture healing. Therefore, we studied the effect of high fat diet-induced (HFD) obesity on callus formation and bone remodelling in a closed femur fracture model in mice. Thirty-one mice were fed a diet containing 60kJ% fat (HFD) for a total of 20 weeks before fracture and during the entire postoperative observation period. Control mice (n=31) received a standard diet containing 10kJ% fat. Healing was analyzed using micro-CT, biomechanical, histomorphometrical, immunohistochemical, serum and protein biochemical analysis at 2 and 4 weeks after fracture. HFD-fed mice showed a higher body weight and increased serum concentrations of leptin and interleukin-6 compared to controls. Within the callus tissue Western blot analyses revealed a higher expression of transcription factor peroxisome proliferator-activated receptor y (PPARy) and a reduced expression of runt-related transcription factor 2 (RUNX2) and bone morphogenetic protein (BMP)-4. However, obesity did not affect the expression of BMP-2 and did not influence the receptor activator of nuclear factor κB (RANK)/RANK ligand/osteoprotegerin (OPG) pathway during fracture healing. Although the bones of HFD-fed animals showed an increased number of adipocytes within the bone marrow, HFD did not increase callus adiposity. In addition, radiological and histomorphometric analysis could also not detect significant differences in bone formation between HFD-fed animals and controls. Accordingly, HFD did not affect bending stiffness after 2 and 4 weeks of healing. These findings indicate that obesity does not affect femur fracture healing in mice.

Effect of Stabilization on the Healing Process of Femur Fractures in Aged Mice.

BACKGROUND: The influence of mechanical stability on fracture healing has previously been studied in adult mice, but is poorly understood in aged animals. Therefore, we herein studied the effect of stabilization on the healing process of femur fractures in aged mice. METHODS: Twenty-four 18-month-old CD-1 mice were stabilized after midshaft fracture of the femur with an intramedullary screw. In another 24 18-month-old mice, the femur fractures were left unstabilized. Bone healing was studied by radiological, biomechanical, histomorphometric, and protein expression analyses. RESULTS: After 2 and 5 weeks of healing, the callus of nonstabilized fractures compared to stabilized fractures was significantly larger, containing a significantly smaller amount of osseous tissue and a higher amount of cartilaginous tissue. This was associated with a significantly lower biomechanical stiffness during the early phase of healing. However, during the late phase of fracture healing both nonstabilized and stabilized fractures showed a biomechanical stiffness of ∼40%. Of interest, Western blot analyses of callus tissue demonstrated that the expression of proteins related to angiogenesis, bone formation and remodeling, i.e. VEGF, CYR61, BMP-2, BMP-4, Col-2, Col-10, RANKL, OPG, did not differ between nonstabilized and stabilized fractures. CONCLUSION: Nonstabilized fractures in aged mice show delayed healing and remodeling. This is not caused by an altered protein expression in the callus but rather by the excessive interfragmentary movements.

Characterization of the healing process in non-stabilized and stabilized femur fractures in mice.

BACKGROUND: Although a variety of suitable fracture models for mice exist, in many studies bone healing was still analyzed without fracture stabilization. Because there is little information whether the healing of non-stabilized fractures differs from that of stabilized fractures, we herein studied the healing process of non-stabilized compared to stabilized femur fractures. MATERIALS AND METHODS: Twenty-one CD-1 mice were stabilized after midshaft fracture of the femur with an intramedullary screw allowing micromovements and endochondral healing. In another 22 mice the femur fractures were left unstabilized. Bone healing was studied by radiological, biomechanical, histomorphometric and protein expression analyses. RESULTS: Non-stabilized femur fractures revealed a significantly lower biomechanical stiffness compared to stabilized fractures. During the early phase of fracture healing non-stabilized fractures demonstrated a significantly lower amount of osseous tissue and a higher amount of cartilage tissue. During the late phase of fracture healing both non-stabilized and stabilized fractures showed almost 100 % osseous callus tissue. However, in stabilized fractures remodeling was almost completed with lamellar bone while non-stabilized fractures still showed large callus with great amounts of woven bone, indicating a delay in bone remodeling. Of interest, western blot analyses of callus tissue demonstrated in non-stabilized fractures a significantly reduced expression of vascular endothelial growth factor and a slightly lowered expression of bone morphogenetic protein-2 and collagen-10. CONCLUSION: Non-stabilized femur fractures in mice show a marked delay in bone healing compared to stabilized fractures. Therefore, non-stabilized fracture models may not be used to analyze the mechanisms of normal bone healing.

Pre-cultivation of adipose tissue-derived microvascular fragments in porous scaffolds does not improve their in vivo vascularisation potential.

Adipose tissue-derived microvascular fragments represent promising vascularisation units for implanted tissue constructs. However, their reassembly into functional microvascular networks takes several days, during which the cells inside the implants are exposed to hypoxia. In the present study, we analysed whether this critical phase may be overcome by pre-cultivation of fragment-seeded scaffolds prior to their implantation. Green fluorescent protein (GFP)-positive microvascular fragments were isolated from epididymal fat pads of male C57BL/6-TgN (ACTB-EGFP) 1Osb/J mice. Nano-size hydroxyapatite particles/poly (ester-urethane) scaffolds were seeded with these fragments and cultivated for 28 days. Subsequently, these scaffolds or control scaffolds, which were freshly seeded with GFP-positive microvascular fragments, were implanted into the dorsal skinfold chamber of C57BL/6 wild-type mice to study their vascularisation and incorporation by means of intravital fluorescence microscopy, histology and immunohistochemistry over 2 weeks. Pre-cultivation of microvascular fragments resulted in the loss of their native vessel morphology. Accordingly, pre-cultivated scaffolds contained a network of individual CD31/GFP-positive endothelial cells with filigrane cell protuberances. After implantation into the dorsal skinfold chamber, these scaffolds exhibited an impaired vascularisation, as indicated by a significantly reduced functional microvessel density and lower fraction of GFP-positive microvessels in their centre when compared to freshly seeded control implants. This was associated with a deteriorated incorporation into the surrounding host tissue. These findings indicate that freshly isolated, non-cultivated microvascular fragments should be preferred as vascularisation units. This would also facilitate their use in clinical practice during intra-operative one-step procedures.

Adipose tissue-derived microvascular fragments from aged donors exhibit an impaired vascularisation capacity.

Adipose tissue-derived microvascular fragments are promising vascularisation units for applications in the field of tissue engineering. Elderly patients are the major future target population of such applications due to an increasing human life expectancy. Therefore, we herein investigated the effect of aging on the fragments' vascularisation capacity. Microvascular fragments were isolated from epididymal fat pads of adult (8 months) and aged (16 months) C57BL/6 donor mice. These fragments were seeded onto porous polyurethane scaffolds, which were implanted into dorsal skinfold chambers to study their vascularisation using intravital fluorescence microscopy, histology and immunohistochemistry. Scaffolds seeded with fragments from aged donors exhibited a significantly lower functional microvessel density and intravascular blood flow velocity. This was associated with an impaired vessel maturation, as indicated by vessel wall irregularities, constantly elevated diameters and a lower fraction of CD31/α-smooth muscle actin double positive microvessels in the implants' border and centre zones. Additional in vitro analyses revealed that microvascular fragments from adult and aged donors do not differ in their stem cell content as well as in their release of angiogenic growth factors, survival and proliferative activity under hypoxic conditions. However, fragments from aged donors exhibit a significantly lower number of matrix metalloproteinase -9-positive perivascular cells. Taken together, these findings demonstrate that aging is a crucial determinant for the vascularisation capacity of isolated microvascular fragments.

Vasopressin induces rectosigmoidal mucosal ischemia during cardiopulmonary bypass.

BACKGROUND: Lower gastrointestinal complications are rare after cardiac surgery with cardiopulmonary bypass (CPB). However, if they occur, they are associated with a high mortality. Endothelin (ET) expression and microcirculatory dysfunction have been shown to be involved in a variety of diseases of the lower gastrointestinal tract. The aim of this study was to analyze whether CPB with or without additional vasopressin administration affects the rectosigmoidal mucosal microcirculation and whether this involves the ET system. METHODS: Pigs were randomized in three groups (n = 6 each): I Sham, II CPB: 1 hour CPB, III CPB + vasopressin: 1 hour CPB and vasopressin (0.006 U/min kg) administration maintaining baseline arterial pressure. All animals were reperfused for 90 minutes. During the experiment hemodynamics and rectosigmoidal mucosal microcirculation were measured continuously. The rectosigmoidal mucosal expression of endothelin-1 (ET-1) and its receptor subtypes A (ETA ) and B (ETB ) were determined using PCR and Western blot analysis. RESULTS: CPB did not change rectosigmoidal microvascular blood flow compared to baseline (68.1 ± 4.0 vs. 75.5 ± 6.6 AU; p = 0.4), but increased ET-1 (gene, 7.8 ± 1.5 vs. 2.3 ± 0.6 RQ; p = 0.002 and protein, 12.0 ± 0.5 vs. 6.9 ± 0.3 OD mm(2) ; p < 0.001), ETA (gene, 2.3 ± 0.6 vs. 0.6 ± 0.1 RQ; p < 0.001 and protein, 11.0 ± 0.3 vs. 6.2 ± 1.1 OD mm(2) ; p = 0.006) and ETB (gene, 6.7 ± 1.2 vs. 1.9 ± 0.3 RQ; p < 0.001 and protein, 25.6 ± 1.4 vs. 14.9 ± 1.5 OD mm(2) ; p = 0.002) expression compared to Sham. Vasopressin during CPB reduced the rectosigmoidal blood flow compared to baseline (26.5 ± 4.9 vs. 75.5 ± 6.6 AU, p < 0.001), and blunted the CPB-induced increase of ET-1 (gene, 1.2 ± 0.4 RQ, p = 0.1 and protein, 8.1 ± 1.6 OD mm(2) , p = 0.5 vs. Sham), ETA (gene, 0.6 ± 0.1 RQ, p = 1.0 and protein, 7.0 ± 0.6 OD mm(2) , p = 0.6 vs. Sham) and ETB (gene, 1.3 ± 0.3 RQ, p = 0.1 and protein, 19.4 ± 2.1 OD mm(2) , p = 0.1 vs. Sham). CONCLUSION: CPB does not significantly affect rectosigmoidal mucosal microcirculation; however, it upregulates ET-1, ETA , and ETB . Vasopressin blunts the CPB-induced elevation of ET-1, ETA , and ETB and induces rectosigmoidal mucosal ischemia during CPB.

Three-dimensional spheroids of adipose-derived mesenchymal stem cells are potent initiators of blood vessel formation in porous polyurethane scaffolds.

Adipose-derived mesenchymal stem cells (adMSCs) exhibit a high angiogenic activity. Accordingly, their incorporation into tissue constructs represents a promising vascularization strategy in tissue engineering. In the present study, we analyzed whether the efficacy of this approach can be improved by seeding adMSCs as three-dimensional spheroids onto porous scaffolds. Green fluorescent protein (GFP)-positive adMSCs expressing CD13, CD73, CD90 and CD117 were isolated from C57BL/6-TgN(ACTB-EGFP)1Osb/J mice for the generation of spheroids using the liquid overlay technique. Porous polyurethane scaffolds were seeded with these spheroids or a comparable number of individual adMSCs and implanted into the dorsal skinfold chamber of C57BL/6 wild-type mice. The vascularization of the implants was analyzed and compared to non-seeded scaffolds by means of intravital fluorescence microscopy and immunohistochemistry. The adMSC spheroids exhibited a homogeneous diameter of ~270µm and could easily be incorporated into the scaffolds by dynamic seeding. After implantation, they induced a strong angiogenic host tissue response, resulting in an improved scaffold vascularization with a significantly higher functional microvessel density when compared to non-seeded scaffolds and scaffolds seeded with individual adMSCs. Immunohistochemical analyses revealed that a high fraction of ~40% of all microvessels within the center of spheroid-seeded scaffolds developed from GFP-positive adMSCs. These vessels inosculated with ingrowing GFP-negative vessels of the host. This indicates that adMSC spheroids serve as individual vascularization units, promoting the simultaneous development of new microvascular networks at different locations inside implanted tissue constructs. Thus, adMSC spheroids may be used to increase the efficacy of MSC-based vascularization strategies in future tissue engineering applications.

Vascularisation of porous scaffolds is improved by incorporation of adipose tissue-derived microvascular fragments.

In tissue engineering, the generation of tissue constructs comprising preformed microvessels is a promising strategy to guarantee their adequate vascularisation after implantation. Herein, we analysed whether this may be achieved by seeding porous scaffolds with adipose tissue-derived microvascular fragments. Green fluorescent protein (GFP)-positive microvascular fragments were isolated by enzymatic digestion from epididymal fat pads of male C57BL/6-TgN(ACTB-EGFP)1Osb/J mice. Nano-size hydroxyapatite particles/poly(ester-urethane) scaffolds were seeded with these fragments and implanted into the dorsal skinfold chamber of C57BL/6 wild-type mice to study inosculation and vascularisation of the implants by means of intravital fluorescence microscopy, histology and immunohistochemistry over 2 weeks. Empty scaffolds served as controls. Vital microvascular fragments could be isolated from adipose tissue and seeded onto the scaffolds under dynamic pressure conditions. In the dorsal skinfold chamber, the fragments survived and exhibited a high angiogenic activity, resulting in the formation of GFP-positive microvascular networks within the implants. These networks developed interconnections to the host microvasculature, resulting in a significantly increased functional microvessel density at day 10 and 14 after implantation when compared to controls. Immunohistochemical analyses of vessel-seeded scaffolds revealed that >90 % of the microvessels in the implants' centre and ~60 % of microvessels in the surrounding host tissue were GFP-positive. This indicates that the scaffolds primarily vascularised by external inosculation. These novel findings demonstrate that the vascularisation of implanted porous scaffolds can be improved by incorporation of microvascular fragments. Accordingly, this approach may markedly contribute to the success of future tissue engineering applications in clinical practice.

Pantoprazole, a proton pump inhibitor, delays fracture healing in mice.

Proton pump inhibitors (PPIs), which are widely used in the treatment of dyspeptic problems, have been shown to reduce osteoclast activity. There is no information, however, on whether PPIs affect fracture healing. We therefore studied the effect of the PPI pantoprazole on callus formation and biomechanics during fracture repair. Bone healing was analyzed in a murine fracture model using radiological, biomechanical, histomorphometric, and protein biochemical analyses at 2 and 5 weeks after fracture. Twenty-one mice received 100 mg/kg body weight pantoprazole i.p. daily. Controls (n = 21) received equivalent amounts of vehicle. In pantoprazole-treated animals biomechanical analysis revealed a significantly reduced bending stiffness at 5 weeks after fracture compared to controls. This was associated with a significantly lower amount of bony tissue within the callus and higher amounts of cartilaginous and fibrous tissue. Western blot analysis showed reduced expression of the bone formation markers bone morphogenetic protein (BMP)-2, BMP-4, and cysteine-rich protein (CYR61). In addition, significantly lower expression of proliferating cell nuclear antigen indicated reduced cell proliferation after pantoprazole treatment. Of interest, the reduced expression of bone formation markers was associated with a significantly diminished expression of RANKL, indicating osteoclast inhibition. Pantoprazole delays fracture healing by affecting both bone formation and bone remodeling.

Xanthohumol inhibits growth and vascularization of developing endometriotic lesions.

BACKGROUND: Xanthohumol is a prenylated flavonoid isolated from hops, which is known to act as a pleiotropic cancer chemopreventive agent owing to its anti-proliferative, anti-inflammatory and anti-angiogenic properties. In the present study, we analyzed, for the first time, whether this dietary compound may also be used for the treatment of endometriosis. METHODS: Peritoneal and mesenteric endometriotic lesions were surgically induced in BALB/c mice by uterine tissue transplantation into the abdominal cavity. The animals were treated daily with 100 µM xanthohumol (n= 8) or vehicle (control, n= 8) via the drinking water, starting 3 days before tissue transplantations. Lesion growth, cyst formation and vascularization were subsequently analyzed by means of high-resolution ultrasound imaging (at Day 0 and then once per week for 28 days), caliper measurements, western blotting, histology and immunohistochemistry over 4 weeks. RESULTS: In the treatment and control groups, uterine grafts developed typical endometriotic lesions with cyst-like dilated glands surrounded by a vascularized endometrial stroma. However, xanthohumol efficiently decreased the size of these lesions at Day 28, independent of their localization within the peritoneal cavity, compared with control (peritoneal: P =0.041; mesenteric: P =0.038). This was associated with a reduced level of phosphoinositide 3-kinase protein. Moreover, vascularization of xanthohumol-treated lesions was suppressed, as indicated by a significantly lower microvessel density at Day 28 when compared with vehicle-treated controls (peritoneal: P =0.026; mesenteric: P =0.004). Additional analyses revealed that treatment with xanthohumol did not affect the histomorphology, proliferation and vascularization of the uterine horns and ovaries. CONCLUSIONS: Taken together, these experimental findings suggest that xanthohumol inhibits the development of endometriotic lesions in mice without inducing serious side effects in the reproductive organs. Thus, xanthohumol represents a promising dietary phytochemical that, after further testing, may be considered for the use in the selective treatment of endometriotic lesions.