In vivo rAAV-mediated human TGF-ß overexpression reduces perifocal osteoarthritis and improves osteochondral repair in a large animal model at one year.

OBJECTIVE: Osteoarthritis (OA) is a serious consequence of focal osteochondral defects. Gene transfer of human transforming growth factor beta (hTGF-ß) with recombinant adeno-associated virus (rAAV) vectors offers a strategy to improve osteochondral repair. However, the long-term in vivo effects of such rAAV-mediated TGF-ß overexpression including its potential benefits on OA development remain unknown. METHOD: Focal osteochondral defects in minipig knees received rAAV-lacZ (control) or rAAV-hTGF-ß in vivo. After one year, osteochondral repair and perifocal OA were visualized using validated macroscopic scoring, ultra-high-field MRI at 9.4 T, and micro-CT. A quantitative estimation of the cellular densities and a validated semi-quantitative scoring of histological and immunohistological parameters completed the analysis of microarchitectural parameters. RESULTS: Direct rAAV-hTGF-ß application induced and maintained significantly improved defect filling and safranin O staining intensity and overall cartilage repair at one year in vivo. In addition, rAAV-hTGF-ß led to significantly higher chondrocyte densities within the cartilaginous repair tissue without affecting chondrocyte hypertrophy and minimized subarticular trabecular separation. Of note, rAAV-hTGF-ß significantly improved the adjacent cartilage structure and chondrocyte density and reduced overall perifocal OA development after one year in vivo. CONCLUSIONS: rAAV-hTGF-ß treatment improves long-term osteochondral repair and delays the progression of perifocal OA in a translational model. These findings have considerable potential for targeted molecular approaches to treat focal osteochondral defects.

Differences in growth and vascularization of ectopic menstrual and non-menstrual endometrial tissue in mouse models of endometriosis.

STUDY QUESTION: Is there a difference in the growth and vascularization between murine endometriotic lesions originating from menstrual or non-menstrual endometrial fragments? SUMMARY ANSWER: Endometriotic lesions developing from menstrual and non-menstrual tissue fragments share many similarities, but also exhibit distinct differences in growth and vascularization, particularly under exogenous estrogen stimulation. WHAT IS KNOWN ALREADY: Mouse models are increasingly used in endometriosis research. For this purpose, menstrual or non-menstrual endometrial fragments serve for the induction of endometriotic lesions. So far, these two fragment types have never been directly compared under identical experimental conditions. STUDY DESIGN, SIZE, DURATION: This was a prospective experimental study in a murine peritoneal and dorsal skinfold chamber model of endometriosis. Endometrial tissue fragments from menstruated (n = 15) and non-menstruated (n = 21) C57BL/6 mice were simultaneously transplanted into the peritoneal cavity or dorsal skinfold chamber of non-ovariectomized (non-ovx, n = 17), ovariectomized (ovx, n = 17) and ovariectomized, estrogen-substituted (ovx+E2, n = 17) recipient animals and analyzed throughout an observation period of 28 and 14 days, respectively. PARTICIPANTS/MATERIALS, SETTING, METHODS: The engraftment, growth and vascularization of the newly developing endometriotic lesions were analyzed by means of high-resolution ultrasound imaging, intravital fluorescence microscopy, histology and immunohistochemistry. MAIN RESULTS AND THE ROLE OF CHANCE: Menstrual and non-menstrual tissue fragments developed into peritoneal endometriotic lesions without differences in growth, microvessel density and cell proliferation in non-ovx mice. Lesion formation out of both fragment types was markedly suppressed in ovx mice. In case of non-menstrual tissue fragments, this effect could be reversed by estrogen supplementation. In contrast, endometriotic lesions originating from menstrual tissue fragments exhibited a significantly smaller volume in ovx+E2 mice, which may be due to a reduced hormone sensitivity. Moreover, menstrual tissue fragments showed a delayed vascularization and a reduced blood perfusion after transplantation into dorsal skinfold chambers when compared to non-menstrual tissue fragments, indicating different vascularization modes of the two fragment types. To limit the role of chance, the experiments were conducted under standardized laboratory conditions. Statistical significance was accepted for a value of P < 0.05. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Endometriotic lesions were induced by syngeneic tissue transplantation into recipient mice without the use of pathological endometriotic tissue of human nature. Therefore, the results obtained in this study may not fully relate to human patients with endometriosis. WIDER IMPLICATIONS OF THE FINDINGS: The present study significantly contributes to the characterization of common murine endometriosis models. These models represent important tools for studies focusing on the basic mechanisms of endometriosis and the development of novel therapeutic strategies for the treatment of this frequent gynecological disease. The presented findings indicate that the combination of different experimental models and approaches may be the most appropriate strategy to study the pathophysiology and drug sensitivity of a complex disease such as endometriosis under preclinical conditions. STUDY FUNDING/COMPETING INTEREST(S): There was no specific funding of this study. The authors have no conflicts of interest to declare.

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.

Seeding density is a crucial determinant for the in vivo vascularisation capacity of adipose tissue-derived microvascular fragments.

Adipose tissue-derived microvascular fragments (ad-MVF) represent effective vascularisation units for the seeding of dermal substitutes. However, particularly in case of extensive skin defects, the required amounts of donor fat tissue for the harvesting of ad-MVF may not always be available. Therefore, we herein determined the lowest ad-MVF density needed to induce a sufficient vascularisation and incorporation of seeded implants. Collagen-glycosaminoglycan matrices (Integra®; diameter: 4 mm) were seeded with 15,000 (HD), 10,000 (MD) and 5,000 (LD) ad-MVF and implanted into full-thickness skin defects within mouse dorsal skinfold chambers, to analyse their in vivo vascularisation and incorporation. Intravital fluorescence microscopy showed a comparable vascularisation of HD and MD ad-MVF-seeded Integra®, which was significantly higher when compared to LD ad-MVF-seeded Integra®. As assessed by photoacoustic imaging, this was associated with an increased oxygenation of the implants. Additional histological and immunohistochemical analyses revealed an enhanced cellular infiltration, collagen content, microvessel density and epithelialisation of HD and MD ad-MVF-seeded Integra®, indicating a better incorporation compared to LD ad-MVF-seeded implants. These findings demonstrate that 80,000 ad-MVF/cm² is the least required density to guarantee an effective vascularisation of the dermal substitute.

Potentials and limitations of Integra® flowable wound matrix seeded with adipose tissue-derived microvascular fragments.

Adipose tissue-derived microvascular fragments (ad-MVF) represent promising vascularisation units for bioengineered Integra® matrix wound dressing (MWD). However, due to the sheet-like structure with small pore sizes, the seeding of this matrix with ad-MVF is mainly limited to its surface. Integra® flowable wound matrix (FWM) may be suitable to achieve a more homogeneous distribution and, thus, improved vascularisation, because this gel-like matrix allows for the direct admixture of ad-MVF during sample preparation. To test this hypothesis, we seeded MWD and FWM with an identical number of ad-MVF and assessed their distribution and inter-fragment distance within both matrices. Moreover, ad-MVF-seeded MWD and FWM were implanted into full-thickness skin defects within mouse dorsal skinfold chambers to analyse their vascularisation, epithelialisation and tissue incorporation using intravital fluorescence microscopy, histology and immunohistochemistry. Seeded FWM exhibited a more homogeneous ad-MVF distribution, when compared to MWD. This resulted in a significantly increased inter-fragment distance, preventing the reassembly of ad-MVF into new microvascular networks. Accordingly, the vascularisation of FWM was diminished after implantation, as indicated by a reduced functional microvessel density and blood perfusion. This was associated with a decreased tissue incorporation and epithelialisation of the matrix, when compared to ad-MVF-seeded MWD. Hence, the use of FWM as a carrier system may require a tremendous amount of ad-MVF to shorten their inter-fragment distance and, thus, to maintain their vascularisation capacity for tissue engineering applications.

IL-17C mediates the recruitment of tumor-associated neutrophils and lung tumor growth.

Chronic obstructive pulmonary disease (COPD) is associated with an increased risk for lung cancer and an aberrant microbiota of the lung. Microbial colonization contributes to chronic neutrophilic inflammation in COPD. Nontypeable Haemophilus influenzae (NTHi) is frequently found in lungs of stable COPD patients and is the major pathogen triggering exacerbations. The epithelial cytokine interleukin-17C (IL-17C) promotes the recruitment of neutrophils into inflamed tissues. The purpose of this study was to investigate the function of IL-17C in the pulmonary tumor microenvironment. We subjected mice deficient for IL-17C (IL-17C-/-) and mice double deficient for Toll-like receptor 2 and 4 (TLR-2/4-/-) to a metastatic lung cancer model. Tumor proliferation and growth as well as the number of tumor-associated neutrophils was significantly decreased in IL-17C-/- and TLR-2/4-/- mice exposed to NTHi. The NTHi-induced pulmonary expression of IL-17C was dependent on TLR-2/4. In vitro, IL-17C increased the NTHi- and tumor necrosis factor-α-induced expression of the neutrophil chemokines keratinocyte-derived chemokine and macrophage inflammatory protein 2 in lung cancer cells but did not affect proliferation. Human lung cancer samples stained positive for IL-17C, and in non-small cell lung cancer patients with lymph node metastasis, IL-17C was identified as a negative prognostic factor. Our data indicate that epithelial IL-17C promotes neutrophilic inflammation in the tumor microenvironment and suggest that IL-17C links a pathologic microbiota, as present in COPD patients, with enhanced tumor growth.

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.

The dorsal skinfold chamber: A versatile tool for preclinical research in tissue engineering and regenerative medicine.

The dorsal skinfold chamber is a rodent model for non-invasive microcirculatory analyses of striated muscle and skin tissue throughout an observation period of 2-3 weeks. In combination with intravital fluorescence microscopy, this model allows the quantitative assessment of dynamic processes such as inflammation, angiogenesis, vascular remodelling and microcirculation. Accordingly, the dorsal skinfold chamber is increasingly used for preclinical research in tissue engineering and regenerative medicine. This includes studies on biocompatibility, vascularisation and incorporation of medical implants and artificial tissue constructs. Moreover, the chamber implantation procedure has been modified to analyse primary and secondary wound healing as well as revascularisation and blood perfusion of dermal substitutes, skin grafts and myocutaneous flaps. Hence, the dorsal skinfold chamber model does not only provide deep insights into fundamental regenerative mechanisms but also represents a versatile tool for the development of novel therapeutic strategies.

Brain damage resulting from postnatal hypoxic-ischemic brain injury is reduced in C57BL/6J mice as compared to C57BL/6N mice.

Perinatal hypoxia is a critical complication during delivery and is mostly studied in animal models of postnatal hypoxic-ischemic brain injury. We here studied the effects of postnatal hypoxic-ischemic brain injury in two different sub-strains of C57BL/6 mice, i.e. C57BL/6J and C57BL/6N mice. These two sub-strains show different metabolic properties, for instance an impaired glucose tolerance in C57BL/6J mice. Genetically, this was linked to differences in their nicotinamide nucleotide transhydrogenase (Nnt) genes: In C57BL/6J mice, exons 7-11 of the Nnt gene are deleted, resulting in the absence of functional Nnt protein. The mitochondrial Nnt-protein is one of several enzymes that catalyses the generation of NADPH, which in turn is important for the elimination of reactive oxygen species (ROS). As ROS is thought to contribute to the pathophysiology of hypoxia-ischemia, the lack of Nnt might indirectly increase ROS levels and therefore result in increased brain damage. We therefore hypothesize that lesion score and lesion size will increase in C57BL/6J mice as compared to C57BL/6N mice. Surprisingly, the results showed exactly the opposite: C57BL/6J mice showed a decrease in lesion score and size, associated with a reduced number of apoptotic cells and activated microglia. In contrast, the number of cells with ROS-induced DNA modifications (detected by 8OHdG) was higher in C57BL/6J than C57BL/6N mice. In conclusion, C57BL/6J mice showed reduced ischemic consequences after postnatal hypoxic-ischemic brain injury compared to C57BL/6N mice, with the exception of the amount of ROS-induced DNA-damage. These differences might relate to the lack of Nnt, but also to a modified metabolic setting (cardiovascular parameters, oxygen and glucose metabolism, immune function) in C57BL/6J mice.

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.

Tissue engineering and regenerative approaches to improving the healing of large bone defects.

Despite the high innate regenerative capacity of bone, large osseous defects fail to heal and remain a clinical challenge. Healing such defects requires the formation of large amounts of bone in an environment often rendered hostile to osteogenesis by damage to the surrounding soft tissues and vasculature. In recent years, there have been intensive research efforts directed towards tissue engineering and regenerative approaches designed to overcome this multifaceted challenge. In this paper, we describe and critically evaluate the state-of-the-art approaches to address the various components of this intricate problem. The discussion includes (i) the properties of synthetic and natural scaffolds, their use in conjunction with cell and growth factor delivery, (ii) their vascularisation, (iii) the potential of gene therapies and (iv) the role of the mechanical environment. In particular, we present a critical analysis of where the field stands, and how it can move forward in a coordinated fashion.

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.

Evaluation of a Gadolinium-Based Nanoparticle (AGuIX) for Contrast-Enhanced MRI of the Liver in a Rat Model of Hepatic Colorectal Cancer Metastases at 9.4 Tesla.

PURPOSE: The aim of this study was to compare a Gd-based nanoparticle (AGuIX) with a standard extracellular Gd-based contrast agent (Gd-DOTA) for MRI at 9.4 T in rats with hepatic colorectal cancer metastases. MATERIALS AND METHODS: 12 rats with hepatic metastases were subjected to MRI using a 9.4 T animal scanner. T1w self-gated FLASH sequences (TR/TE = 45/2.5 ms, alpha = 45°, TA = 1: 23 min, FOV = 5.12 × 5.12 cm(2), matrix = 256 × 256) were acquired before and at 10 time points after contrast injection. Each animal received 0.1 mmol/kg BW Gd-DOTA i.v. 2 days later AGuIX was applied at 0.01 mmol/kg BW (representing equal Gd doses). The SNR of normal liver (SNRliver), hyper- and hypoenhancing parts of tumors (SNRtumor, hyperenh/SNRtumor, hypoenhanc), erector spinae muscle (SNRmuscle), CNR and lesion enhancement (LE) were calculated based on ROI measurements. RESULTS: Mean SNRliver (Gd-DOTA: 14.6 +/- 0.7; AGuIX: 28.2+/- 2.6, p < 0.001), SNRtumor, hyperenhanc (Gd-DOTA: 18.6 +/- 1.2; AGuIX: 29.6 +/- 2.8, p < 0.001), SNRtumor, hypoenhanc (Gd-DOTA: 12.0 +/- 0.7; AGuIX: 15.4 +/- 0.7, p < 0.001), SNRmuscle (Gd-DOTA: 12.3 +/- 0.3; AGuIX: 14.0 +/- 0.7, p < 0.001), mean CNR (Gd-DOTA: -2.5 +/- 0.2; AGuIX: -7.5 +/- 1.0, p < 0.001) and LE (Gd-DOTA: 3.8 +/- 0.7; AGuIX: 14.9 +/- 2.8, p = 0.001) were significantly higher using AGuIX. Regardless of the larger molecular size, AGuIX demonstrates an early peak enhancement followed by a continuous washout. CONCLUSION: AGuIX provides better enhancement at 9.4 T compared to Gd-DOTA for equal doses of applied Gd. This is based on the molecule structure and the subsequent increased interaction with protons leading to a higher relaxivity. AGuIX potentially ameliorates the conspicuity of focal liver lesions and may improve the sensitivity in diagnostic imaging of malignant hepatic tumors. KEY POINTS: AGuIX provides superior enhancement as compared to the extracellular compound Gd-DOTA at 9.4 T. AGuIX may improve the detection and diagnostic sensitivity of malignant focal liver lesions. The small size of AGuIX allows for fast renal clearance and prevents undesirable accumulation in the body.