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.

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.

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.

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.

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.

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.

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.

Endometriosis: Survey of Current Diagnostic and Therapeutic Options and Latest Research Work.

Endometriosis is one of the most frequent benign diseases in women of child-bearing age. The main symptoms are chronic upper abdominal pain and infertility. However, the aetiology and pathogenesis of endometriosis are as yet insufficiently clarified. Thus, therapy is mainly symptomatic with laparoscopic surgery being the gold standard. The aim of drug therapy is to achieve a hypo-oestrogenic condition. In cases of severe endometriosis and a desire to have children there is often an indication for assisted reproduction. The present article illustrates almost all current aspects on the diagnosis of and therapy of endometriosis. From the clinical viewpoint, emphasis is placed on the rare cases of deeply infiltrating endometriosis that are, however, accompanied with a high morbidity. Current therapeutic options in cases of infertility are also presented in more detail. Furthermore, special attention is paid to the latest research results from both clinical and basic research fields in order to demonstrate our current knowledge on the pathogenesis and, where possible, potentially related therapeutic options.

Locally applied macrophage-activating lipopeptide-2 (MALP-2) promotes early vascularization of implanted porous polyethylene (Medpor®).

Porous polyethylene (Medpor®) is frequently used in craniofacial reconstructive surgery. Rapid vascularization of the biomaterial crucially contributes to its adequate incorporation without complications. Macrophage-activating lipopeptide-2 (MALP-2) is a toll-like receptor (TLR)-2/6 agonist with pro-angiogenic properties. Herein we analyzed whether local single-shot application of MALP-2 improves the angiogenic host tissue response to Medpor®. Medpor® (3 mm×3 mm×0.25 mm) was implanted into dorsal skinfold chambers of BALB/c mice topically exposed to different MALP-2 doses (0.1 and 0.5 µg) or vehicle (control). The vascularization of the implants and the inflammatory foreign body reaction was analyzed using intravital fluorescence microscopy, histology and immunohistochemistry over 14 days. MALP-2 treatment dose-dependently improved the vascularization of Medpor®, as indicated by a significantly higher functional microvessel density at the border and center of the implants when compared to controls. This was associated with a temporary increase of adherent leukocytes in host tissue venules during the first 3 days after implantation. At day 14, implants in MALP-2-treated chambers were surrounded by granulation tissue, which exhibited a significantly higher density of CD31-positive microvessels and number of F4/80-positive macrophages when compared to controls. Additional biomaterial-free chambers did not show any signs of angiogenesis when treated with MALP-2. This indicates that locally applied MALP-2 effectively stimulates the early vascularization of Medpor® without inducing any local or systemic side effects. Accordingly, this easy approach may further improve the rapid incorporation of this biomaterial at the implantation site.

Cell therapy for bone repair: narrowing the gap between vision and practice.

This position paper summarises a vision of how cell-based therapies can be applied clinically to regenerate bone, as well as the steps needed to narrow the gap between that vision and clinical reality. It is a result of the presentations and discussion of the "Cell Therapy for Bone Repair" breakout session at the AO Foundation Symposium "Where Science Meets Clinics" in Davos, Switzerland from September 5-7, 2013. Participants included leaders from science, medicine, and industry from around the world. The session included clinical and scientific presentations, as well as an extended discussion among participants. Bone tissue has an innate regenerative capacity that in most cases allows functional healing at damage sites. However, there are a number of serious conditions in which bone does not fully heal and the result is significant morbidity. The clinical need for new therapies is clear, and the breakout session participants were enthusiastic about the potential impact on cell-based therapies for bone repair in the clinic. However, they also recognised the significant challenges that face the development of commercially viable cell therapy products. This paper outlines a vision in which patient selection is based on expected therapeutic outcome to create a consistently successful, cost-effective, cell-based therapy for bone repair. The need for a more complete understanding of bone repair, a better infrastructure for preclinical studies, and the need for collaboration among stakeholders is discussed.

Early host tissue response to different types of vascular prostheses coated with silver acetate or vaporized metallic silver.

OBJECTIVES: In vascular surgery, the infection of prosthetic vascular grafts represents a serious life-threatening complication. Due to the increasing resistance of hospital micro-organisms to standard antibiotic therapies, maximum effort should be put in the primary prevention of such infections. For this purpose, grafts may be coated with different antibacterial silver formulations. In the present study the different effects of silver acetate-coating and vaporized metallic silver-coating on the vascularization and perigraft inflammation during the initial phase after implantation of Intergard Silver (IS) and Silver Graft (SG) were compared. METHODS: Silver acetate-coated IS and vaporized metallic silver-coated SG were implanted into the dorsal skinfold chamber of C57BL/6 mice (n = 8 per group) to study angiogenesis and leukocyte inflammation at the implantation site by means of repetitive intravital fluorescence microscopy over a 14-day period. At the end of the in vivo experiments, apoptosis and cell proliferation in the newly developed granulation tissue surrounding the implants was analyzed by immunohistochemistry. RESULTS: IS exhibited an improved vascularization, resulting in a significantly higher functional capillary density when compared to SG. Moreover, the leukocyte inflammatory response to IS was less pronounced, as indicated by a reduced number of adherent leukocytes in perigraft venules. This was associated with a higher proliferative activity of the granulation tissue incorporating the IS when compared to SG. The numbers of apoptotic cells in the perigraft tissue were low and did not differ between the two groups. CONCLUSION: Silver acetate-coated IS exhibits an improved vascularization and reduced perigraft inflammation during the first 14 days after implantation when compared to vaporized metallic silver-coated SG. This may contribute to reducing the risk of early perigraft seroma formation and subsequent infection.

Combined blockade of angiotensin II type 1 receptor and activation of peroxisome proliferator-activated receptor-γ by telmisartan effectively inhibits vascularization and growth of murine endometriosis-like lesions.

STUDY QUESTION: Is telmisartan effective in the treatment of endometriosis? SUMMARY ANSWER: Combined blockade of angiotensin II type 1 receptor (AT1R) and activation of peroxisome proliferator-activated receptor (PPAR)-γ by telmisartan inhibits vascularization and growth of murine endometriosis-like lesions. WHAT IS KNOWN ALREADY: AT1R and PPAR-γ are involved in the regulation of inflammation, proliferation and angiogenesis. These processes are also crucial for the pathogenesis of endometriosis and both receptors are expressed in endometrial tissue. Telmisartan is a partial agonist of PPAR-γ, which additionally blocks AT1R. STUDY DESIGN, SIZE, DURATION: This was a randomized study in the mouse dorsal skinfold chamber and peritoneal model of endometriosis. Endometriosis-like lesions were induced in dorsal skinfold chambers of 21 female C57BL/6 mice, and in the peritoneal cavity of 15 additional animals, which were daily treated with an i.p. injection of pioglitazone (10 mg/kg, n = 12), telmisartan (10 mg/kg, n = 12) or vehicle (5% dimethyl sulfoxide (DMSO), n = 12) throughout an observation period of 14 and 28 days, respectively. PARTICIPANTS/MATERIALS, SETTING, METHODS: The anti-angiogenic actions of pioglitazone, a full PPAR-γ agonist, and telmisartan were firstly assessed in vitro by an aortic ring assay. Endometriosis-like lesions were induced in the dorsal skinfold chamber or peritoneal cavity and the effects of telmisartan and pioglitazone on their vascularization, immune cell content and growth were studied by intravital fluorescence microscopy, high-resolution ultrasound imaging as well as histological, immunohistochemical and immunofluorescent analyses. Additional quantitative real-time polymerase chain reaction (qRT-PCR) arrays served for gene expression profiling of the lesions. To limit the role of chance, the experiments were conducted under standardized laboratory conditions with appropriate vehicle-treated controls. Statistical significance was accepted for a value of P < 0.05. MAIN RESULTS AND THE ROLE OF CHANCE: Telmisartan inhibited vascular sprout formation of aortic rings more effectively than pioglitazone. Accordingly, endometriosis-like lesions in dorsal skinfold chambers of telmisartan-treated animals exhibited a markedly lower functional microvessel density and blood perfusion. High-resolution ultrasound analyses of peritoneal endometriosis-like lesions revealed that the compound inhibited the stromal tissue growth, resulting in a significantly reduced final lesion volume. In contrast, the development of cysts did not differ between the groups. Moreover, telmisartan induced an up-regulation of PPAR-γ and a down-regulation of AT1R proteins in endometriosis-like lesions, which was associated with a decreased density of CD31-positive microvessels, a reduced immune cell content and a lower number of Ki67-positive proliferating cells. qRT-PCR arrays further demonstrated an inhibitory action of telmisartan on the expression of several angiogenic and inflammatory genes. LIMITATIONS, REASONS FOR CAUTION: Endometriosis-like 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: This study demonstrates that telmisartan inhibits vascularization, immune cell content and growth of endometriosis-like lesions. Accordingly, the combined blockade of AT1R and activation of PPAR-γ represents a promising new concept in the development of novel compounds for the treatment of endometriosis. STUDY FUNDING/COMPETING INTEREST(S): There was no specific funding of this study. The authors have no conflicts of interest to declare.

The plasma protein fibrinogen stabilizes clusters of red blood cells in microcapillary flows.

The supply of oxygen and nutrients and the disposal of metabolic waste in the organs depend strongly on how blood, especially red blood cells, flow through the microvascular network. Macromolecular plasma proteins such as fibrinogen cause red blood cells to form large aggregates, called rouleaux, which are usually assumed to be disaggregated in the circulation due to the shear forces present in bulk flow. This leads to the assumption that rouleaux formation is only relevant in the venule network and in arterioles at low shear rates or stasis. Thanks to an excellent agreement between combined experimental and numerical approaches, we show that despite the large shear rates present in microcapillaries, the presence of either fibrinogen or the synthetic polymer dextran leads to an enhanced formation of robust clusters of red blood cells, even at haematocrits as low as 1%. Robust aggregates are shown to exist in microcapillaries even for fibrinogen concentrations within the healthy physiological range. These persistent aggregates should strongly affect cell distribution and blood perfusion in the microvasculature, with putative implications for blood disorders even within apparently asymptomatic subjects.