Cilostazol Stimulates Angiogenesis and Accelerates Fracture Healing in Aged Male and Female Mice by Increasing the Expression of PI3K and RUNX2.

Fracture healing in the aged is associated with a reduced healing capacity, which often results in delayed healing or non-union formation. Many factors may contribute to this deterioration of bone regeneration, including a reduced 'angiogenic trauma response'. The phosphodiesterase-3 (PDE-3) inhibitor cilostazol has been shown to exert pro-angiogenic and pro-osteogenic effects in preclinical studies. Therefore, we herein analyzed in a stable closed femoral fracture model whether this compound also promotes fracture healing in aged mice. Forty-two aged CD-1 mice (age: 16-18 months) were daily treated with 30 mg/kg body weight cilostazol (n = 21) or vehicle (control, n = 21) by oral gavage. At 2 and 5 weeks after fracture, the femora were analyzed by X-ray, biomechanics, micro-computed tomography (µCT), histology, immunohistochemistry, and Western blotting. These analyses revealed a significantly increased bending stiffness at 2 weeks (2.2 ± 0.4 vs. 4.3 ± 0.7 N/mm) and an enhanced bone formation at 5 weeks (4.4 ± 0.7 vs. 9.1 ± 0.7 mm3) in cilostazol-treated mice when compared to controls. This was associated with a higher number of newly formed CD31-positive microvessels (3.3 ± 0.9 vs. 5.5 ± 0.7 microvessels/HPF) as well as an elevated expression of phosphoinositide-3-kinase (PI3K) (3.6 ± 0.8 vs. 17.4 ± 5.5-pixel intensity × 104) and runt-related transcription factor (RUNX)2 (6.4 ± 1.2 vs. 18.2 ± 2.7-pixel intensity × 104) within the callus tissue. These findings indicate that cilostazol accelerates fracture healing in aged mice by stimulating angiogenesis and the expression of PI3K and RUNX2. Hence, cilostazol may represent a promising compound to promote bone regeneration in geriatric patients.

Parathyroid hormone stimulates bone regeneration in an atrophic non-union model in aged mice.

BACKGROUND: Non-union formation still represents a major burden in trauma and orthopedic surgery. Moreover, aged patients are at an increased risk for bone healing failure. Parathyroid hormone (PTH) has been shown to accelerate fracture healing in young adult animals. However, there is no information whether PTH also stimulates bone regeneration in atrophic non-unions in the aged. Therefore, the aim of the present study was to analyze the effect of PTH on bone regeneration in an atrophic non-union model in aged CD-1 mice. METHODS: After creation of a 1.8 mm segmental defect, mice femora were stabilized by pin-clip fixation. The animals were treated daily with either 200 mg/kg body weight PTH 1-34 (n = 17) or saline (control; n = 17) subcutaneously. Bone regeneration was analyzed by means of X-ray, biomechanics, micro-computed tomography (µCT) imaging as well as histological, immunohistochemical and Western blot analyses. RESULTS: In PTH-treated animals bone formation was markedly improved when compared to controls. This was associated with an increased bending stiffness as well as a higher number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts and CD31-positive microvessels within the callus tissue. Furthermore, PTH-treated aged animals showed a decreased inflammatory response, characterized by a lower number of MPO-positive granulocytes and CD68-positive macrophages within the bone defects when compared to controls. Additional Western blot analyses demonstrated a significantly higher expression of cyclooxygenase (COX)-2 and phosphoinositide 3-kinase (PI3K) in PTH-treated mice. CONCLUSION: Taken together, these findings indicate that PTH is an effective pharmacological compound for the treatment of non-union formation in aged animals.

Sildenafil, a phosphodiesterase-5 inhibitor, stimulates angiogenesis and bone regeneration in an atrophic non-union model in mice.

Non-union formation represents a major complication in trauma and orthopedic surgery. The phosphodiesterase-5 (PDE-5) inhibitor sildenafil has been shown to exert pro-angiogenic and pro-osteogenic effects in vitro and in vivo. Therefore, the aim of the present study was to analyze the impact of sildenafil in an atrophic non-union model in mice. After creation of a 1.8 mm segmental defect, mice femora were stabilized by pin-clip fixation. Bone regeneration was analyzed by means of X-ray, biomechanics, photoacoustic and micro-computed tomography (µCT) imaging as well as histological, immunohistochemical and Western blot analyses at 2, 5 and 10 weeks after surgery. The animals were treated daily with either 5 mg/kg body weight sildenafil (n = 35) or saline (control; n = 35) per os. Bone formation was markedly improved in defects of sildenafil-treated mice when compared to controls. This was associated with a higher bending stiffness as well as an increased number of CD31-positive microvessels and a higher oxygen saturation within the callus tissue. Moreover, the bone defects of sildenafil-treated animals contained more tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts and CD68-positive macrophages and exhibited a higher expression of the pro-angiogenic and pro-osteogenic markers cysteine rich protein (CYR)61 and vascular endothelial growth factor (VEGF) when compared to controls. These findings demonstrate that sildenafil acts as a potent stimulator of angiogenesis and bone regeneration in atrophic non-unions.

Cilostazol improves hepatic blood perfusion, microcirculation, and liver regeneration after major hepatectomy in rats.

Major hepatectomy or small-for-size liver transplantation may result in postoperative liver failure. So far, no treatment is available to improve liver regeneration. Herein, we studied whether cilostazol, a selective phosphodiesterase III inhibitor, is capable of improving liver regeneration after major hepatectomy. Sprague-Dawley rats (n = 74) were treated with cilostazol (5 mg/kg daily) or a glucose solution and underwent either 70% liver resection or a sham operation. Before and after surgery, hepatic arterial and portal venous blood flow and hepatic microvascular perfusion were analyzed. Liver morphology, function, and regeneration were studied with histology, immunohistochemistry, western blotting, and bile excretion analysis. Cilostazol significantly increased hepatic blood flow and microcirculation before and after hepatectomy in comparison with sham-operated controls. This was associated with an elevation of hepatic vascular endothelial growth factor expression, an increase of hepatocellular proliferation, and an acceleration of liver regeneration. Furthermore, cilostazol protected the tissue of the remnant liver as indicated by an attenuation of hepatocellular disintegration. In conclusion, cilostazol increases hepatic blood perfusion, microcirculation, and liver regeneration after a major hepatectomy. Thus, cilostazol may represent a novel strategy to reduce the rate of liver failure after both extended hepatectomy and small-for-size liver transplantation.

Sildenafil delays bone remodeling of fractured femora in aged mice by reducing the number and activity of osteoclasts within the callus tissue.

The elderly exhibit a reduced healing capacity after fracture, which is often associated with delayed or failed bone healing. This is due to a plethora of factors, such as an impaired bone vascular system and delayed angiogenesis. The phosphodiesterase-5 (PDE-5) inhibitor sildenafil exerts pro-angiogenic and pro-osteogenic effects. Hence, we herein investigated in aged mice whether sildenafil can improve fracture healing. For this purpose, 40 aged CD-1 mice (16-18 months) were daily treated with 5 mg/kg body weight sildenafil (n = 20) or vehicle (control, n = 20) by oral gavage. The callus tissue of their femora was analyzed at 2 and 5 weeks after fracture by X-ray, biomechanics, micro-computed tomography (µCT), histology, immunohistochemistry as well as Western blotting. These analyses revealed a significantly increased bone volume and higher ratio of callus to femoral bone diameter in sildenafil-treated mice at 5 weeks after fracture when compared to controls. This was associated with a reduced number and activity of osteoclasts at 2 weeks after fracture, most likely caused by an increased expression of osteoprotegerin (OPG). Taken together, these findings indicate that sildenafil does not improve fracture healing in the elderly but delays the process of bone remodeling most likely by reducing the number and activity of osteoclasts within the callus tissue.

Long-term preconditioning with erythropoietin reduces ischemia-induced skin necrosis.

OBJECTIVE: Recent findings have attested to EPO tissue-protective effects in ischemically challenged tissues. Therefore, the study aimed at elaborating the effect of systemic pre- and postconditioning using EPO in a mouse model of persistent ischemia of the skin. METHODS: Three groups of nine C57Bl/6-mice each were analyzed. The experimental groups consisted of untreated controls, EPO preconditioning, and EPO postconditioning (500 IU EPO/kg bw/day for 10 days). Critically perfused skin flaps undergoing necrosis, if kept untreated, were mounted into dorsal skinfold chambers. Intravital epi-fluorescence microscopy was performed for 10 days to assess tissue necrosis, microcirculation, inflammation, and angiogenesis. Protein expression analysis of eNOS was performed. Hematocrit analyses were carried out separately in eight animals. RESULTS: Only EPO preconditioning was able to significantly reduce necrosis, when compared with controls. This correlated with a significantly increased CD in the critically perfused tissue. Administration of EPO only slightly increased eNOS expression at day 10, when compared with controls. EPO induced angiogenesis and increased hematocrit. Finally, EPO significantly reduced leukocytic inflammation in arterioles in all EPO receiving mice. CONCLUSIONS: EPO preconditioning effectively reduces skin necrosis predominantly by capillary maintenance and reperfusion, as well as improved tissue regeneration. Thus, EPO preconditioning might represent a promising, non-invasive approach to reduce complications in ischemically challenged skin.

Inhibition of angiogenesis: a novel antitumor mechanism of the herbal compound arctigenin.

Arctigenin, a functional ingredient of several traditional Chinese herbs, has been reported to have potential antitumor activity. However, its mechanisms of action are still not well elucidated. Because the establishment and metastatic spread of tumors is crucially dependent on angiogenesis, here we investigated whether arctigenin inhibits tumor growth by disturbing blood vessel formation. For this purpose, human dermal microvascular endothelial cells were exposed to different arctigenin doses to study their viability, proliferation, protein expression, migration, and tube formation compared with vehicle-treated controls. In addition, arctigenin action on vascular sprouting was analyzed in an aortic ring assay. Furthermore, we studied direct arctigenin effects on CT26.WT colon carcinoma cells. Spheroids of these tumor cells were transplanted into the dorsal skinfold chamber of arctigenin-treated and vehicle-treated BALB/c mice for the in-vivo analysis of tumor vascularization and growth by intravital fluorescence microscopy, histology, and immunohistochemistry. We found that noncytotoxic doses of arctigenin dose dependently reduced the proliferation of human dermal microvascular endothelial cells without affecting their migratory and tube-forming capacity. Arctigenin treatment also resulted in a decreased cellular expression of phosphorylated serine/threonine protein kinase AKT, vascular endothelial growth factor receptor 2, and proliferating cell nuclear antigen and inhibited vascular sprouting from aortic rings. In addition, proliferation, but not secretion of vascular endothelial growth factor, was decreased in arctigenin-treated tumor cells. Finally, arctigenin suppressed the vascularization and growth of engrafting CT26.WT tumors in the dorsal skinfold chamber model. Taken together, these results show for the first time an antiangiogenic action of arctigenin, which may contribute considerably toward its antitumor activity.

Hepatic arterial infusion but not systemic application of cetuximab in combination with oxaliplatin significantly reduces growth of CC531 colorectal rat liver metastases.

PURPOSE: Systemic chemotherapy still represents the gold standard in the treatment of irresectable colorectal liver metastases. Modern anticancer agents like the monoclonal antibody cetuximab have improved the outcome of patients in clinical studies. As hepatic arterial infusion (HAI) is capable to potentially increase the anticancer effect of cytostatics, we herein studied whether HAI of cetuximab (CE) as a single agent or in combination with oxaliplatin (OX) exerts increased anticancer effects compared to the systemic application (SYS) of the drugs. METHODS: WAG/Rij rats were randomized to eight groups and underwent 10 days after subcapsular hepatic tumor implantation either HAI or SYS of CE, OX, or the combination of both agents (CE + OX). Saline-treated animals served as controls. Tumor volume was measured at days 10 and 13 using three-dimensional ultrasound. On day 13, liver and tumor tissue was sampled for histological and immunohistochemical analysis. RESULTS: In controls, the tumor volume significantly increased from day 10 to 13. Application of OX alone via HAI or SYS did not inhibit tumor growth compared to controls. SYS of CE or CE + OX did also not reduce tumor growth. In contrast, HAI of CE and CE + OX significantly inhibited tumor growth. HAI of CE significantly reduced tumor vascularization as measured by the number of platelet endothelial cell adhesion molecule-1-positive cells and significantly increased the number of apoptotic tumor cells as measured by the cellular caspase-3 expression. CONCLUSION: HAI of CE and CE + OX reduces tumor growth of colorectal rat liver metastases involving the inhibition of angiogenesis and induction of tumor cell apoptosis.

Cilostazol promotes blood vessel formation and bone regeneration in a murine non-union model.

Non-unions represent a major complication in trauma and orthopedic surgery. Many factors contribute to bone regeneration, out of which an adequate vascularization has been recognized as crucial. The phosphodiesterase-3 (PDE-3) inhibitor cilostazol has been shown to exert pro-angiogenic and pro-osteogenic effects in a variety of preclinical studies. Hence, we herein investigated the effects of cilostazol on bone regeneration in an atrophic non-union model in mice. For this purpose, a 1.8 mm femoral segmental defect was stabilized by pin-clip fixation and the animals were treated daily with 30 mg/kg body weight cilostazol or saline (control) per os. At 2, 5 and 10 weeks after surgery the healing of femora was analyzed by X-ray, biomechanics, photoacoustic imaging, and micro-computed tomography (µCT). To investigate the cellular composition and the growth factor expression of the callus tissue additional histological, immunohistochemical and Western blot analyses were performed. Cilostazol-treated animals showed increased bone formation within the callus, resulting in an enhanced bending stiffness when compared to controls. This was associated with a more pronounced expression of vascular endothelial growth factor (VEGF), a higher number of CD31-positive microvessels and an increased oxygen saturation within the callus tissue. Furthermore, cilostazol induced higher numbers of tartrate-resistant acidic phosphate (TRAP)-positive osteoclasts and CD68-positive macrophages. Taken together, these findings demonstrate that cilostazol is a promising drug candidate for the adjuvant treatment of atrophic non-unions in clinical practice.

Diclofenac, a NSAID, delays fracture healing in aged mice.

Nonsteroidal anti-inflammatory drugs (NSAIDs), such as diclofenac, belong to the most prescribed analgesic medication after traumatic injuries. However, there is accumulating evidence that NSAIDs impair fracture healing. Because bone regeneration in aged patients is subject to significant changes in cell differentiation and proliferation as well as a markedly altered pharmacological action of drugs, we herein analyzed the effects of diclofenac on bone healing in aged mice using a stable closed femoral facture model. Thirty-three mice (male n = 14, female n = 19) received a daily intraperitoneal injection of diclofenac (5 mg/kg body weight). Vehicle-treated mice (n = 29; male n = 13, female n = 16) served as controls. Fractured mice femora were analyzed by means of X-ray, biomechanics, micro computed tomography (µCT), histology and Western blotting. Biomechanical analyses revealed a significantly reduced bending stiffness in diclofenac-treated animals at 5 weeks after fracture when compared to vehicle-treated controls. Moreover, the callus tissue in diclofenac-treated aged animals exhibited a significantly reduced amount of bone tissue and higher amounts of fibrous tissue. Further histological analyses demonstrated less lamellar bone after diclofenac treatment, indicating a delay in callus remodeling. This was associated with a decreased number of osteoclasts and an increased expression of osteoprotegerin (OPG) during the early phase of fracture healing. These findings indicate that diclofenac delays fracture healing in aged mice by affecting osteogenic growth factor expression and bone formation as well as osteoclast activity and callus remodeling.

Endothelial progenitor cells contribute to the vascularization of endometriotic lesions.

Endometriosis is a frequent gynecological disease that is characterized by the development of vascularized endometriotic lesions inside the peritoneal cavity. Herein, we analyzed whether circulating endothelial progenitor cells (EPCs) are recruited and incorporated into the microvasculature of these lesions. Intraperitoneal endometriotic lesions were surgically induced in irradiated FVB/N mice, which were reconstituted with bone marrow from FVB/N-TgN (Tie2/green fluorescent protein [GFP]) 287 Sato mice. Vascularization and recruitment of GFP-positive EPCs in the lesions was analyzed by intravital fluorescence microscopy and immunohistochemistry over 4 weeks. The numbers of stem cell antigen-1 (Sca-1)/vascular endothelial growth factor receptor-2-positive EPCs in blood and hematopoietic organs of additional endometriotic and control mice were assessed by flow cytometry. We found that approximately 15% of the microvascular endothelium in engrafting endometriotic lesions consisted of incorporated GFP-positive EPCs. Recruitment of EPCs into the lesions coincided with the establishment of own blood supply and the expression of stromal cell-derived factor-1. Accordingly, treatment with the stromal cell-derived factor-1/chemokine receptor type 4 axis antagonist AMD3100 significantly decreased the number of recruited EPCs and the vascularization of endometriotic lesions. However, endometriosis did not induce increased levels of EPCs in the blood, bone marrow, and spleen of C57BL/6 mice. To our knowledge, our findings indicate for the first time that vasculogenesis (ie, de novo generation of blood vessels from EPCs) may represent an integral mechanism in the pathogenesis of endometriosis.

Increased osteoblast and osteoclast activity in female senescence-accelerated, osteoporotic SAMP6 mice during fracture healing.

BACKGROUND: Previous studies have shown that fracture healing depends on gender and that in females, ovariectomy-induced osteoporosis impairs the healing process. There is no information, however, whether the alteration of fracture healing in osteoporosis also depends on gender. MATERIALS AND METHODS: Therefore, we herein studied fracture healing in female and male senescence-accelerated osteoporotic mice, strain P6 (SAMP6), including biomechanical, histomorphometric, and protein biochemical analysis. RESULTS: Bending stiffness was reduced in male and female SAMP6 mice compared with senescence-resistant strain 1 (SAMR1) controls. This was associated with elevated serum concentrations of tartrate-resistent acid phosphatase form 5b (TRAP) in both female and male SAMP6 mice. Callus size, however, was significantly larger in female SAMP6 mice compared with male SAMP6 mice and female SAMR1 controls. This indicates a delayed remodeling process in female SAMP6 mice. The delay of callus remodeling in female SAMP6 mice was associated with a significantly higher osteoprotegerin (OPG) callus tissue expression and increased serum concentrations of osteocalcin (OC) and deoxypyridinoline (DPD), indicating elevated osteoblast and osteoclast activities. CONCLUSION: The present study shows that remodeling during fracture healing in female, but not in male, SAMP6 mice is delayed, most probably due to an increased osteoblast and osteoclast activity.

Melatonin impairs fracture healing by suppressing RANKL-mediated bone remodeling.

BACKGROUND: Melatonin, the major pineal hormone, is known to regulate distinct physiologic processes. Previous studies have suggested that it supports skeletal growth and bone formation, most probably by inhibiting bone resorption. There is no information, however, whether melatonin affects fracture healing. We therefore studied in a mouse femur fracture model the influence of melatonin on callus formation and biomechanics during fracture healing. METHODS AND MATERIALS: Thirty CD-1 mice received 50 mg/kg body weight melatonin i.p. daily during the entire 2-wk or 5-wk observation period. Controls (n = 30) received equivalent amounts of vehicle. Bone healing was studied by radiological, biomechanical, histomorphometrical, and protein biochemical analyses at 2 and 5 wk after fracture. RESULTS: Biomechanical analysis at 2 wk after fracture healing showed a significantly lower bending stiffness in melatonin-treated animals compared with controls. A slightly higher amount of cartilage tissue and a significantly larger callus size indicated a delayed remodeling process after melatonin treatment. Western blot analysis showed a significantly reduced expression of receptor activator of nuclear factor-κB ligand (RANKL) and collagen I after melatonin treatment. The reduced expression of RANKL was associated with a diminished number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts within the callus of the newly formed bone. CONCLUSIONS: Because bone resorption is an essential requirement for adequate remodeling during fracture healing, we conclude that melatonin impairs fracture healing by suppressing bone resorption through down-regulation of RANKL-mediated osteoclast activation.

Indole-3-Carbinol Inhibits the Growth of Endometriotic Lesions by Suppression of Microvascular Network Formation.

Endometriosis represents an estrogen-dependent disorder with a complex pathophysiology. Phytochemicals are promising candidates for endometriosis therapy, because they simultaneously target different cellular processes involved in the pathogenesis of endometriosis. Herein, we analyzed whether indole-3-carbinol (I3C) suppresses the development of endometriotic lesions, which were surgically induced by fixation of uterine tissue samples (diameter: 2 mm) from female BALB/c donor mice to the peritoneum of recipient animals. The mice received either I3C or vehicle (control) by peroral administration once per day. Growth, cyst formation, cell proliferation, microvascularization and protein expression of the lesions were assessed by high-resolution ultrasound imaging, caliper measurements, histology, immunohistochemistry and Western blotting. I3C inhibited the vascularization and growth of endometriotic lesions without inducing anti-angiogenic and anti-proliferative side effects on reproductive organs. This was associated with a significantly reduced number of proliferating stromal and endothelial cells and a lower expression of the pro-angiogenic signaling molecules vascular endothelial growth factor receptor-2 (VEGFR2), phosphoinositide 3-kinase (PI3K) and phosphorylated extracellular signal-regulated kinase (pERK) within I3C-treated lesions when compared to controls. These findings indicate that I3C effectively inhibits endometriotic lesion formation in mice. Thus, further studies should clarify whether I3C may be also beneficial for the prevention and therapy of the human disease.

The ischemic time window of ectopic endometrial tissue crucially determines its ability to develop into endometriotic lesions.

Endometriosis develop from shed endometrial fragments via retrograde menstruation. This affects the survival, proliferation and vascularization of the tissue and its final ability to form endometriotic lesions. Within this study, uterine tissue samples from donor mice were precultivated for 24 h or 72 h to simulate avascular periods. Their morphology, microvessel density, apoptotic activity and expression of angiogenesis-related proteins were analyzed in vitro. The formation of endometriotic lesions in vivo was assessed after transplantation of precultivated uterine tissue samples to the abdominal wall and dorsal skinfold chambers by means of high-resolution ultrasound, intravital fluorescence microscopy, histology and immunohistochemistry. In vitro, 72-h-precultivated uterine tissue samples exhibit extensive areas of tissue necrosis and high numbers of apoptotic cells as well as a significantly reduced cell and microvessel density. These samples failed to develop into endometriotic lesions. In contrast, the 24-h-precultivated samples showed, that their early vascularization and growth in vivo was improved when compared to controls. This indicates that avascular periods have a strong impact on the survival of ectopic endometrial tissue and the chance for the development of endometriosis.

Comparison of two non-union models with damaged periosteum in mice: Segmental defect and pin-clip fixation versus transverse fracture and K-wire stabilization.

Despite growing knowledge about the mechanisms of fracture healing, non-union formation still represents a major complication in trauma and orthopedic surgery. Non-union models in mice gain increasing interest, because they allow investigating the molecular and cellular mechanisms of failed fracture healing. These models often use segmental defects to achieve non-union formation. Alternatively, failed fracture healing can be induced by transverse fractures with additional periosteal injury. The present study systematically compared the reliability of these two approaches to serve as non-union model. A 0.6 mm K-wire was inserted into the femora of CD-1 mice in a retrograde fashion and a closed transverse femoral fracture was created. Subsequently, the fracture site was exposed and the periosteum was cauterized. This approach was compared with a well-established non-union model involving the pin-clip fixation of a 1.8 mm segmental defect. The callus tissue was analyzed by means of radiography, biomechanics, histology and Western blotting. At 10 weeks after surgery 10 out of 12 femora (83.3 %) of the K-wire group showed a non-union formation. The pin-clip model resulted in 100 % non-union formation. The K-wire group showed increased bone formation, osteoclast activity and bending stiffness when compared to the group with pin-clip fixation. This was associated with a higher expression of bone formation markers. However, the number of CD31-positive microvessels was reduced in the K-wire group, indicating an impaired angiogenic capacity after periosteal cauterization. These findings suggest that the pin-clip model is more reliable for the study of non-union formation in mice. The K-wire model including periosteal injury by cauterization however, may be particularly applied in preclinical studies which explore the effects of damaged periosteum and reduced angiogenic capacity to trauma-induced fractures.

Macrophage-activating lipoprotein (MALP)-2 impairs the healing of partial tendon injuries in mice.

Tendon injuries are accounted for up to 50% of musculoskeletal injuries and often result in poor outcomes. Inflammation is a major hallmark of tendon regeneration. Therefore, we analyzed in this study whether the topical application of the pro-inflammatory mediator macrophage-activating lipoprotein (MALP)-2 improves the healing of partial tendon injuries. C57BL/6 mice underwent a partial tenotomy of the flexor digitorum longus tendon of the left hind limb, which was treated with a solution containing either 0.5 µg MALP-2 or vehicle (control). Repetitive gait analyses were performed prior to the surgical intervention as well as postoperatively on days 1, 3, 7, 14 and 36. The structural stability of the tendons was biomechanically tested on day 7 and 36. In addition, Western blot analyses were performed on isolated tendons that were treated in vitro with MALP-2 or vehicle. In both groups, partial tenotomy resulted in a pathological gait pattern during the initial postoperative phase. On day 7, the gait pattern normalized in vehicle-treated animals, but not in MALP-2-treated mice. Moreover, the tendons of MALP-2-treated mice exhibited a significantly reduced biomechanical stiffness after 7 and 36 days when compared to controls. Western blot analyses revealed a significantly higher expression of heme oxygenase (HO)-1 and lower expression of cyclin D in MALP-2-treated tendons. These findings indicate that MALP-2 delays the healing of injured tendons most likely due to increased intracellular stress and suppressed cell proliferation in this naturally bradytrophic tissue. Hence, the application of MALP-2 cannot be recommended for the treatment of tendon injuries.

Adipose tissue-derived microvascular fragments promote lymphangiogenesis in a murine lymphedema model.

Chronic lymphedema after cancer treatment is common and there is still no cure for this disease. We herein investigated the lymphangiogenic capacity of adipose tissue-derived microvascular fragments (MVF), which contain stem cells and lymphatic vessel fragments. Secondary lymphedema was induced in the hindlimbs of C57BL/6J mice. Green fluorescence protein (GFP)+ MVF were isolated from transgenic C57BL/6Tg (CAG-EGFP)1Osb/J mice, suspended in collagen hydrogel, and injected in the lymphadenectomy defect of wild-type animals. This crossover model allowed the detection of MVF-derived blood and lymphatic vessels after transplantation. The MVF group was compared with animals receiving collagen hydrogel only or a sham intervention. Lymphangiogenic effects were analyzed using volumetry, magnetic resonance (MR) lymphography, histology, and immunohistochemistry. MVF injection resulted in reduced hindlimb volumes when compared to non-treated controls. MR lymphography revealed lymphatic regeneration with reduced dermal backflow after MVF treatment. Finally, MVF transplantation promoted popliteal angiogenesis and lymphangiogenesis associated with a significantly increased microvessel and lymphatic vessel density. These findings indicate that MVF transplantation represents a promising approach to induce therapeutic lymphangiogenesis.

Establishment of a Reliable Model to Study the Failure of Fracture Healing in Aged Mice.

The failure of fracture healing represents a substantial clinical problem. Moreover, aged patients demonstrate an elevated risk for failed bone healing. However, murine models to study the failure of fracture healing are established only in young adult animals. Therefore, the aim of this study was to develop a reliable model to study failed fracture healing in aged mice. After creation of a 1.8-mm segmental defect and periosteal resection, femora of aged mice (18-20 months) and young adult control mice (3-4 months) were stabilized by pin-clip fixation. Segmental defects were analyzed by means of biomechanics, x-ray, and micro-computed tomography, as well as histomorphometric, immunohistochemical, and Western blot analysis. After 10 weeks, all animals showed a complete lack of osseous bridging, resulting in fracture healing failure. Segmental defects in aged mice revealed a reduced bone formation and vascularization when compared to young adult mice. This was associated with a decreased expression of bone formation markers. In addition, we detected a reduced number of tartrate-resistant acid phosphatase-positive osteoclasts and an elevated osteoprotegerin/receptor activator of NF-ĸB ligand ratio in aged animals, indicating a reduced osteoclast activity. Moreover, aged animals showed also an enhanced inflammatory response, characterized by an increased infiltration of macrophages within the callus tissue. Taken together, we herein report for the first time a reliable model to study fracture healing failure in aged mice. In the future, the use of this model enables us to study novel therapeutic strategies and molecular mechanics of failed fracture healing during aging.