53 research outputs found
Macrophages promote network formation and maturation of transplanted adipose tissue-derived microvascular fragments
Adipose tissue-derived microvascular fragments rapidly reassemble into microvascular networks within implanted scaffolds. Herein, we analyzed the contribution of macrophages to this process. C57BL/6 mice received clodronate (clo)-containing liposomes for macrophage depletion, whereas animals treated with phosphate-buffered-saline-containing liposomes served as controls. Microvascular fragments were isolated from clo- and phosphate-buffered-saline-treated donor mice and seeded onto collagen-glycosaminoglycan matrices, which were implanted into dorsal skinfold chambers of clo- and phosphate-buffered-saline-treated recipient mice. The implants' vascularization and incorporation were analyzed by stereomicroscopy, intravital fluorescence microscopy, histology, and immunohistochemistry. Compared to controls, matrices within clo-treated animals exhibited a significantly reduced functional microvessel density. Moreover, they contained a lower fraction of microvessels with an α-smooth muscle actin (SMA)+ cell layer, indicating impaired vessel maturation. This was associated with a deteriorated implant incorporation. These findings demonstrate that macrophages not only promote the reassembly of microvascular fragments into microvascular networks, but also improve their maturation during this process
Erythropoietin exposure of isolated pancreatic islets accelerates their revascularization after transplantation
Aims
The exposure of isolated pancreatic islets to pro-angiogenic factors prior to their transplantation represents a promising strategy to accelerate the revascularization of the grafts. It has been shown that erythropoietin (EPO), a glycoprotein regulating erythropoiesis, also induces angiogenesis. Therefore, we hypothesized that EPO exposure of isolated islets improves their posttransplant revascularization.
Methods
Flow cytometric, immunohistochemical and quantitative real-time (qRT)-PCR analyses were performed to study the effect of EPO on the viability, cellular composition and gene expression of isolated islets. Moreover, islets expressing a mitochondrial or cytosolic H2O2 sensor were used to determine reactive oxygen species (ROS) levels. The dorsal skinfold chamber model in combination with intravital fluorescence microscopy was used to analyze the revascularization of transplanted islets.
Results
We found that the exposure of isolated islets to EPO (3 units/mL) for 24 h does not affect the viability and the production of ROS when compared to vehicle-treated and freshly isolated islets. However, the exposure of islets to EPO increased the number of CD31-positive cells and enhanced the gene expression of insulin and vascular endothelial growth factor (VEGF)-A. The revascularization of the EPO-cultivated islets was accelerated within the initial phase after transplantation when compared to both controls.
Conclusion
These findings indicate that the exposure of isolated islets to EPO may be a promising approach to improve clinical islet transplantation
Darbepoetin-α increases the blood volume flow in transplanted pancreatic islets in mice
Aims
The minimal-invasive transplantation of pancreatic islets is a promising approach to treat diabetes mellitus type 1. However, islet transplantation is still hampered by the insufficient process of graft revascularization, leading to a poor clinical outcome. Accordingly, the identification of novel compounds, which accelerate and improve the revascularization of transplanted islets, is of great clinical interest. Previous studies have shown that darbepoetin (DPO)-α, a long lasting analogue of erythropoietin, is capable of promoting angiogenesis. Hence, we investigated in this study whether DPO improves the revascularization of transplanted islets.
Methods
Islets were isolated from green fluorescent protein-positive FVB/N donor mice and transplanted into dorsal skinfold chambers of FVB/N wild-type animals, which were treated with DPO low dose (2.5 ”g/kg), DPO high dose (10 ”g/kg) or vehicle (control). The revascularization was assessed by repetitive intravital fluorescence microscopy over an observation period of 14 days. Subsequently, the cellular composition of the grafts was analyzed by immunohistochemistry.
Results
The present study shows that neither low- nor high-dose DPO treatment accelerates the revascularization of free pancreatic islet grafts. However, high-dose DPO treatment increased the blood volume flow of the transplanted islet.
Conclusions
These findings demonstrated that DPO treatment does not affect the revascularization of transplanted islets. However, the glycoprotein increases the blood volume flow of the grafts, which results in an improved microvascular function and may facilitate successful transplantation
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 efect 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 fxation. The animals
were treated daily with either 200 mg/kg body weight PTH 1â34 (n=17) or saline (control; n=17) subcutaneously.
Bone regeneration was analyzed by means of X-ray, biomechanics, micro-computed tomography (”CT) 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 stifness 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 infammatory 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 signifcantly higher expression of cyclooxygenase (COX)-2 and phosphoinositide 3-kinase
(PI3K) in PTH-treated mice.
Conclusion Taken together, these fndings indicate that PTH is an efective pharmacological compound for the treatment of non-union formation in aged animals
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). Vehicletreated 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 (ÎŒCT), 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
Erythropoietin accelerates the revascularization of transplanted pancreatic islets
Background and Purpose
Pancreatic islet transplantation is a promising therapeutic approach for Type 1 diabetes. A major prerequisite for the survival of grafted islets is a rapid revascularization after transplantation. Erythropoietin (EPO), the primary regulator of erythropoiesis, has been shown to promote angiogenesis. Therefore, we investigated in this study whether EPO improves the revascularization of transplanted islets.
Experimental Approach
Islets from FVB/N mice were transplanted into dorsal skinfold chambers of recipient animals, which were daily treated with an intraperitoneal injection of EPO (500 IU·kgâ1) or vehicle (control) throughout an observation period of 14 days. In a second set of experiments, animals were only pretreated with EPO over a 6âday period prior to islet transplantation. The revascularization of the grafts was assessed by repetitive intravital fluorescence microscopy and immunohistochemistry. In addition, a streptozotocinâinduced diabetic mouse model was used to study the effect of EPOâpretreatment on the endocrine function of the grafts.
Key Results
EPO treatment slightly accelerated the revascularization of the islet grafts. This effect was markedly more pronounced in EPOâpretreated animals, resulting in significantly higher numbers of engrafted islets and an improved perfusion of endocrine tissue without affecting systemic haematocrit levels when compared with controls. Moreover, EPOâpretreatment significantly accelerated the recovery of normoglycaemia in diabetic mice after islet transplantation.
Conclusion and Implications
These findings demonstrate that, particularly, shortâterm EPOâpretreatment represents a promising therapeutic approach to improve the outcome of islet transplantation, without an increased risk of thromboembolic events
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 (”CT). 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
Bone Healing Gone Wrong : Pathological Fracture Healing and Non-UnionsâOverview of Basic and Clinical Aspects and Systematic Review of Risk Factors
Bone healing is a multifarious process involving mesenchymal stem cells, osteoprogenitor
cells, macrophages, osteoblasts and -clasts, and chondrocytes to restore the osseous tissue. Particularly
in long bones including the tibia, clavicle, humerus and femur, this process fails in 2â10% of all
fractures, with devastating effects for the patient and the healthcare system. Underlying reasons for
this failure are manifold, from lack of biomechanical stability to impaired biological host conditions
and wound-immanent intricacies. In this review, we describe the cellular components involved in
impaired bone healing and how they interfere with the delicately orchestrated processes of bone
repair and formation. We subsequently outline and weigh the risk factors for the development of
non-unions that have been established in the literature. Therapeutic prospects are illustrated and put
into clinical perspective, before the applicability of biomarkers is finally discussed
Radiographic, Biomechanical and Histological Characterization of Femoral Fracture Healing in Aged CD-1 Mice
With a gradually increasing elderly population, the treatment of geriatric patients represents
a major challenge for trauma and reconstructive surgery. Although, it is well established that aging
affects bone metabolism, it is still controversial if aging impairs bone healing. Accordingly, we
investigated fracture healing in young adult (3â4 months) and aged (16â18 months) CD-1 mice using
a stable closed femoral fracture model. Bone healing was analyzed by radiographic, biomechanical
and histological analysis at 1, 2, 3, 4 and 5 weeks after fracture. Our results demonstrated an increased
callus diameter to femoral diameter ratio in aged animals at later time points of fracture healing
when compared to young adult mice. Moreover, our biomechanical analysis revealed a significantly
decreased bending stiffness at 3 and 4 weeks after fracture in aged animals. In contrast, at 5 weeks
after fracture, the analysis showed no significant difference in bending stiffness between the two study
groups. Additional histological analysis showed a delayed endochondral ossification in aged animals
as well as a higher amounts of fibrous tissue at early healing time points. These findings indicate a
delayed process of callus remodeling in aged CD-1 mice, resulting in a delayed fracture healing when
compared to young adult animals. However, the overall healing capacity of the fractured femora was
not affected by aging
Pantoprazole impairs fracture healing in aged mice
Proton pump inhibitors (PPIs) belong to the most common medication in geriatric medicine. They are known to reduce osteoclast activity and to delay fracture healing in young adult mice. Because differentiation and proliferation in fracture healing as well as pharmacologic actions of drugs markedly differ in the elderly compared to the young, we herein studied the effect of the PPI pantoprazole on bone healing in aged mice using a murine fracture model. Bone healing was analyzed by biomechanical, histomorphometric, radiological and protein biochemical analyses. The biomechanical analysis revealed a significantly reduced bending stiffness in pantoprazole-treated animals when compared to controls. This was associated with a decreased amount of bone tissue within the callus, a reduced trabecular thickness and a higher amount of fibrous tissue. Furthermore, the number of osteoclasts in pantoprazole-treated animals was significantly increased at 2Â weeks and decreased at 5Â weeks after fracture, indicating an acceleration of bone turnover. Western blot analysis showed a lower expression of the bone morphogenetic protein-4 (BMP-4), whereas the expression of the pro-angiogenic parameters was higher when compared to controls. Thus, pantoprazole impairs fracture healing in aged mice by affecting angiogenic and osteogenic growth factor expression, osteoclast activity and bone formation
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