CK2 activity is crucial for proper glucagon expression.

AIMS/HYPOTHESIS: Protein kinase CK2 acts as a negative regulator of insulin expression in pancreatic beta cells. This action is mainly mediated by phosphorylation of the transcription factor pancreatic and duodenal homeobox protein 1 (PDX1). In pancreatic alpha cells, PDX1 acts in a reciprocal fashion on glucagon (GCG) expression. Therefore, we hypothesised that CK2 might positively regulate GCG expression in pancreatic alpha cells. METHODS: We suppressed CK2 kinase activity in αTC1 cells by two pharmacological inhibitors and by the CRISPR/Cas9 technique. Subsequently, we analysed GCG expression and secretion by real-time quantitative RT-PCR, western blot, luciferase assay, ELISA and DNA pull-down assays. We additionally studied paracrine effects on GCG secretion in pseudoislets, isolated murine islets and human islets. In vivo, we examined the effect of CK2 inhibition on blood glucose levels by systemic and alpha cell-specific CK2 inhibition. RESULTS: We found that CK2 downregulation reduces GCG secretion in the murine alpha cell line αTC1 (e.g. from 1094±124 ng/l to 459±110 ng/l) by the use of the CK2-inhibitor SGC-CK2-1. This was due to a marked decrease in Gcg gene expression through alteration of the binding of paired box protein 6 (PAX6) and transcription factor MafB to the Gcg promoter. The analysis of the underlying mechanisms revealed that both transcription factors are displaced by PDX1. Ex vivo experiments in isolated murine islets and pseudoislets further demonstrated that CK2-mediated reduction in GCG secretion was only slightly affected by the higher insulin secretion after CK2 inhibition. The kidney capsule transplantation model showed the significance of CK2 for GCG expression and secretion in vivo. Finally, CK2 downregulation also reduced the GCG secretion in islets isolated from humans. CONCLUSIONS/INTERPRETATION: These novel findings not only indicate an important function of protein kinase CK2 for proper GCG expression but also demonstrate that CK2 may be a promising target for the development of novel glucose-lowering drugs.

Locally Directed Recombinant Adeno- Associated Virus-Mediated IGF-1 Gene Therapy Enhances Osteochondral Repair and Counteracts Early Osteoarthritis In Vivo.

BACKGROUND: Restoration of osteochondral defects is critical, because osteoarthritis (OA) can arise. HYPOTHESIS: Overexpression of insulin-like growth factor 1 (IGF-1) via recombinant adeno-associated viral (rAAV) vectors (rAAV-IGF-1) would improve osteochondral repair and reduce parameters of early perifocal OA in sheep after 6 months in vivo. STUDY DESIGN: Controlled laboratory study. METHODS: Osteochondral defects were created in the femoral trochlea of adult sheep and treated with rAAV-IGF-1 or rAAV-lacZ (control) (24 defects in 6 knees per group). After 6 months in vivo, osteochondral repair and perifocal OA were assessed by well-established macroscopic, histological, and immunohistochemical scoring systems as well as biochemical and micro-computed tomography evaluations. RESULTS: Application of rAAV-IGF-1 led to prolonged (6 months) IGF-1 overexpression without adverse effects, maintaining a significantly superior overall cartilage repair, together with significantly improved defect filling, extracellular matrix staining, cellular morphology, and surface architecture compared with rAAV-lacZ. Expression of type II collagen significantly increased and that of type I collagen significantly decreased. Subchondral bone repair and tidemark formation were significantly improved, and subchondral bone plate thickness and subarticular spongiosa mineral density returned to normal. The OA parameters of perifocal structure, cell cloning, and matrix staining were significantly better preserved upon rAAV-IGF-1 compared with rAAV-lacZ. Novel mechanistic associations between parameters of osteochondral repair and OA were identified. CONCLUSION: Local rAAV-mediated IGF-1 overexpression enhanced osteochondral repair and ameliorated parameters of perifocal early OA. CLINICAL RELEVANCE: IGF-1 gene therapy may be beneficial in repair of focal osteochondral defects and prevention of perifocal OA.

Modulation of early osteoarthritis by tibiofemoral re-alignment in sheep.

OBJECTIVE: To investigate whether tibiofemoral alignment influences early knee osteoarthritis (OA). We hypothesized that varus overload exacerbates early degenerative osteochondral changes, and that valgus underload diminishes early OA. METHOD: Normal, over- and underload were induced by altering alignment via high tibial osteotomy in adult sheep (n = 8 each). Simultaneously, OA was induced by partial medial anterior meniscectomy. At 6 weeks postoperatively, OA was examined in five individual subregions of the medial tibial plateau using Kellgren-Lawrence grading, quantification of macroscopic OA, semiquantitative histopathological OA and immunohistochemical type-II collagen, ADAMTS-5, and MMP-13 scoring, biochemical determination of DNA and proteoglycan contents, and micro-computed tomographic evaluation of the subchondral bone. RESULTS: Multivariate analyses revealed that OA cartilaginous changes had a temporal priority over subchondral bone changes. Underload inhibited early cartilage degeneration in a characteristic topographic pattern (P ≥ 0.0983 vs. normal), in particular below the meniscal damage, avoided alterations of the subarticular spongiosa (P ≥ 0.162 vs. normal), and prevented the disturbance of otherwise normal osteochondral correlations. Overload induced early alterations of the subchondral bone plate microstructure towards osteopenia, including significantly decreased percent bone volume and increased bone surface-to-volume ratio (all P ≤ 0.0359 vs. normal). CONCLUSION: The data provide high-resolution evidence that tibiofemoral alignment modulates early OA induced by a medial meniscus injury in adult sheep. Since underload inhibits early OA, these data also support the clinical value of strategies to reduce the load in an affected knee compartment to possibly decelerate structural OA progression.

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.

Short-Term Periodic Fasting Reduces Ischemia-Induced Necrosis in Musculocutaneous Flap Tissue.

Periodic fasting (PF) as a form of dietary restriction has been shown to induce tissue-protective effects against ischemic injury in several different tissues. Accordingly, in this study we analyzed whether a short-term 24 h fast is suitable to prevent necrosis of musculocutaneous flap tissue undergoing acute persistent ischemia. C57BL/6N mice were randomly divided into a PF group (n = 8) and a control group that was given unrestricted access to standard chow (n = 8). The PF animals underwent a 24 h fast immediately before flap elevation and had unrestricted access to food for the rest of the 10 day observation period. Musculocutaneous flaps with a random pattern design were dissected on the animals' backs and mounted into dorsal skinfold chambers. On days 1, 3, 5, 7 and 10 after surgery, nutritive tissue perfusion, angiogenesis and flap necrosis were evaluated using intravital fluorescence microscopy. Thereafter, the flap tissue was excised and fixed for histological and immunohistochemical analyses. The flaps of PF-treated animals exhibited a higher functional capillary density and more newly formed microvessels, resulting in a significantly increased flap survival rate. Moreover, they contained a lower number of myeloperoxidase (MPO)-positive neutrophilic granulocytes and cleaved caspase-3-positive apoptotic cells in the transition zone between vital and necrotic flap tissue. These findings indicate that short-term PF improves tissue survival in ischemically challenged musculocutaneous flaps by maintaining nutritive blood perfusion and dampening ischemia-induced inflammation.

Microvascular Fragment-Loaded Platelet-Rich Plasma Dressing Promotes Cutaneous Wound Healing.

Objective: Chronic wounds represent a considerable burden for the affected patients and the health care system. To overcome this problem, effective treatment strategies are urgently required. In this study, we tested a novel approach by combining platelet-rich plasma (PRP) and microvascular fragments (MVF) to create a prevascularized gel dressing. Approach: MVF were enzymatically isolated from the epididymal fat pads of transgenic green fluorescent protein (GFP)+ C57BL/6J donor mice. Subsequently, 5,000 MVF were suspended in 10 µL murine PRP as carrier and transferred into full-thickness skin wounds within dorsal skinfold chambers of C57BL/6J wild-type mice (PRP+MVF). Wound healing in comparison to empty wounds (control) and wounds filled with PRP alone was repeatedly analyzed throughout 14 days by means of stereomicroscopy, histology, and immunohistochemistry. Results: Planimetric assessment of the wound size over time revealed a significantly accelerated and improved healing of PRP+MVF-treated wounds when compared with PRP-treated and empty control wounds. These wounds also exhibited a significantly higher density of blood and lymph vessels, which originated from the GFP+ MVF isolates and effectively promoted granulation tissue formation inside the skin defects. Innovation: This study is the first to combine PRP and MVF for the improvement of wound healing. Conclusion: The combination of PRP and MVF represents a promising approach for the future treatment of wounds that do not heal spontaneously due to poor wound-healing conditions.

C-Myc/H19/miR-29b axis downregulates nerve/glial (NG)2 expression in glioblastoma multiforme.

Nerve/glial antigen (NG)2 is highly expressed in glioblastoma multiforme (GBM). However, the underlying mechanisms of its upregulated expression are largely unknown. In silico analyses reveal that the tumor-suppressive miR-29b targets NG2. We used GBM-based data from The Cancer Genome Atals databases to analyze the expression pattern of miR-29b and different target genes, including NG2. Moreover, we investigated the regulatory function of miR-29b on NG2 expression and NG2-related signaling pathways. We further studied upstream mechanisms affecting miR-29b-dependent NG2 expression. We found that miR-29b downregulates NG2 expression directly and indirectly via the transcription factor Sp1. Furthermore, we identified the NG2 coreceptor platelet-derived growth factor receptor (PDGFR)α as an additional miR-29b target. As shown by a panel of functional cell assays, a reduced miR-29b-dependent NG2 expression suppresses tumor cell proliferation and migration. Signaling pathway analyses revealed that this is associated with a decreased ERK1/2 activity. In addition, we found that the long noncoding RNA H19 and c-Myc act as upstream repressors of miR-29b in GBM cells, resulting in an increased NG2 expression. These findings indicate that the c-Myc/H19/miR-29b axis crucially regulates NG2 expression in GBM and, thus, represents a target for the development of future GBM therapies.

Nanofat Accelerates and Improves the Vascularization, Lymphatic Drainage and Healing of Full-Thickness Murine Skin Wounds.

The treatment of wounds using the body's own resources is a promising approach to support the physiological regenerative process. To advance this concept, we evaluated the effect of nanofat (NF) on wound healing. For this purpose, full-thickness skin defects were created in dorsal skinfold chambers of wild-type mice. These defects were filled with NF generated from the inguinal subcutaneous adipose tissue of green fluorescent protein (GFP)+ donor mice, which was stabilized using platelet-rich plasma (PRP). Empty wounds and wounds solely filled with PRP served as controls. Wound closure, vascularization and formation of granulation tissue were repeatedly analyzed using stereomicroscopy, intravital fluorescence microscopy, histology and immunohistochemistry over an observation period of 14 days. PRP + NF-treated wounds exhibited accelerated vascularization and wound closure when compared to controls. This was primarily due to the fact that the grafted NF contained a substantial fraction of viable GFP+ vascular and lymph vessel fragments, which interconnected with the GFP- vessels of the host tissue. Moreover, the switch from inflammatory M1- to regenerative M2-polarized macrophages was promoted in PRP + NF-treated wounds. These findings indicate that NF markedly accelerates and improves the wound healing process and, thus, represents a promising autologous product for future wound management.

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.

Immunomodulatory Effects of Histone Variant H2A.J in Ionizing Radiation Dermatitis.

PURPOSE: Histone variant H2A.J is associated with premature senescence after ionizing radiation (IR) and modulates senescence-associated secretory phenotype (SASP). Using constitutive H2A.J knock-out mice, the role of H2A.J was investigated in radiation dermatitis. METHODS AND MATERIALS: H2A.J wild-type (WT) and knock-out (KO) mice were exposed to moderate or high IR doses (≤20 Gy, skinfold IR). Radiation-induced skin reactions were investigated up to 2 weeks post-IR at macroscopic and microscopic levels. H2A.J and other senescence markers, as well as DNA damage and proliferation markers, were studied by immunohistochemistry, immunofluorescence, and electron microscopy. After high-dose IR, protein-coding transcriptomes were analyzed by RNA sequencing, immune cell infiltration by flow cytometry, and gene expression by reverse transcription polymerase chain reaction in (non-) irradiated WT versus KO skin. RESULTS: In WT skin, epidermal keratinocytes showed time- and dose-dependent H2A.J accumulation after IR exposure. Unexpectedly, stronger inflammatory reactions with increased epidermal thickness and progressive hair follicle loss were observed in irradiated KO versus WT skin. Clearly more radiation-induced senescence was observed in keratinocyte populations of KO skin after moderate and high doses, with hair follicle stem cells being particularly badly damaged, leading to follicle atrophy. After high-dose IR, transcriptomic analysis revealed enhanced senescence-associated signatures in irradiated KO skin, with intensified release of SASP factors. Flow cytometric analysis indicated increased immune cell infiltration in both WT and KO skin; however, specific chemokine-mediated signaling in irradiated KO skin led to more neutrophil recruitment, thereby aggravating radiation toxicities. Increased skin damage in irradiated KO skin led to hyperproliferation, abnormal differentiation, and cornification of keratinocytes, accompanied by increased upregulation of transcription-factor JunB. CONCLUSIONS: Lack of radiation-induced H2A.J expression in keratinocytes is associated with increased senescence induction, modulation of SASP expression, and exacerbated inflammatory skin reactions. Hence, epigenetic H2A.J-mediated gene expression in response to IR regulates keratinocyte immune functions and plays an essential role in balancing the inflammatory response during radiation dermatitis.

A cell-free, biomimetic hydrogel based on probiotic membrane vesicles ameliorates wound healing.

Probiotic bacteria, such as Lactobacilli, have been shown to elicit beneficial effects in various tissue regeneration applications. However, their formulation as living bacteria is challenging, and their therapeutic use as proliferating microorganisms is especially limited in immunocompromised patients. Here, we propose a new therapeutic avenue to circumvent these shortcomings by developing a bacteriomimetic hydrogel based on membrane vesicles (MVs) produced by Lactobacilli. We coupled MVs from Lactobacillus plantarum and Lactobacillus casei, respectively, to the surface of synthetic microparticles, and embedded those bacteriomimetics into a pharmaceutically applicable hydrogel matrix. The wound microenvironment changes during the wound healing process, including adaptions of the pH and changes of the oxygen supply. We thus performed proteomic characterization of the MVs harvested under different culture conditions and identified characteristic proteins related to the biological effect of the probiotics in every culture state. In addition, we highlight a number of unique proteins expressed and sorted into the MVs for every culture condition. Using different in vitro models, we demonstrated that increased cell migration and anti-inflammatory effects of the bacteriomimetic microparticles were dependent on the culture condition of the secreting bacteria. Finally, we demonstrated the bacteriomimetic hydrogel's ability to improve healing in an in vivo mouse full-thickness wound model. Our results create a solid basis for the future application of probiotic-derived vesicles in the treatment of inflammatory dispositions and stimulates the initiation of further preclinical trials.

Actin-templated Structures: Nature's Way to Hierarchical Surface Patterns (Gecko's Setae as Case Study).

The hierarchical design of the toe pad surface in geckos and its reversible adhesiveness have inspired material scientists for many years. Micro- and nano-patterned surfaces with impressive adhesive performance have been developed to mimic gecko's properties. While the adhesive performance achieved in some examples has surpassed living counterparts, the durability of the fabricated surfaces is limited and the capability to self-renew and restore function-inherent to biological systems-is unimaginable. Here the morphogenesis of gecko setae using skin samples from the Bibron´s gecko (Chondrodactylus bibronii) is studied. Gecko setae develop as specialized apical differentiation structures at a distinct cell-cell layer interface within the skin epidermis. A primary role for F-actin and microtubules as templating structural elements is necessary for the development of setae's hierarchical morphology, and a stabilization role of keratins and corneus beta proteins is identified. Setae grow from single cells in a bottom layer protruding into four neighboring cells in the upper layer. The resulting multicellular junction can play a role during shedding by facilitating fracture of the cell-cell interface and release of the high aspect ratio setae. The results contribute to the understanding of setae regeneration and may inspire future concepts to bioengineer self-renewable patterned adhesive surfaces.

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.

Generation of Connective Tissue-Free Microvascular Fragment Isolates from Subcutaneous Fat Tissue of Obese Mice.

BACKGROUND: Microvascular fragment (MVF) isolates are generated by short-term enzymatic digestion of adipose tissue and contain numerous vessel segments for the vascularization of tissue defects. Recent findings indicate that the functionality of these isolates is determined by the quality of the fat source. Therefore, we compared MVF isolates from subcutaneous adipose tissue of obese and lean mice. METHODS: MVF isolates were generated from subcutaneous adipose tissue of donor mice, which received a high fat or control diet for 12 weeks. The isolates were analyzed in vitro and in vivo. RESULTS: Feeding of mice with a high fat diet induced obesity with adipocyte hypertrophy, resulting in a significantly lower collagen fraction and microvessel density within the subcutaneous fat depots when compared to lean controls. Accordingly, MVF isolates from obese mice also contained a reduced number of MVF per mL adipose tissue. However, these MVF tended to be longer and, in contrast to MVF from lean mice, were not contaminated with collagen fibers. Hence, they could be freely seeded onto collagen-glycosaminoglycan scaffolds, whereas MVF from lean controls were trapped in between large amounts of collagen fibers that clogged the pores of the scaffolds. In line with these results, scaffolds seeded with MVF isolates from obese mice exhibited a significantly improved in vivo vascularization after implantation into full-thickness skin defects. CONCLUSION: Subcutaneous adipose tissue from obese mice facilitates the generation of connective tissue-free MVF isolates. Translated to clinical conditions, these findings suggest that particularly obese patients may benefit from MVF-based vascularization strategies.

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.

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.

Caloric Restriction: A Novel Conditioning Strategy to Improve the Survival of Ischemically Challenged Musculocutaneous Random Pattern Flaps.

Caloric restriction (CR) is a cost-effective and easy-to-perform approach to counteracting surgical stress. The present study therefore evaluates the tissue-protective effects of a 30% CR in musculocutaneous flaps undergoing ischemia. For this purpose, a well-established murine dorsal skinfold chamber model, in combination with random pattern musculocutaneous flaps, was used. C57BL/6N mice were divided at random into a CR group (n = 8) and a control group with unrestricted access to standard chow (n = 8). The CR animals were subjected to a 30% reduction in caloric intake for 10 days before flap elevation. Intravital fluorescence microscopy was carried out on days 1, 3, 5, 7 and 10 after flap elevation to assess the nutritive blood perfusion, angiogenesis and flap necrosis. Subsequently, the flap tissue was harvested for additional histological and immunohistochemical analyses. The CR-treated animals exhibited a significantly higher functional capillary density and more newly formed microvessels within the flap tissue when compared to the controls; this was associated with a significantly higher flap survival rate. Immunohistochemical analyses showed a decreased invasion of myeloperoxidase-positive neutrophilic granulocytes into the flap tissue of the CR-treated mice. Moreover, the detection of cleaved caspase-3 revealed fewer cells undergoing apoptosis in the transition zone between the vital and necrotic tissue in the flaps of the CR-treated mice. These results demonstrate that a CR of 30% effectively prevents flap necrosis by maintaining microperfusion on a capillary level and inhibiting inflammation under ischemic stress. Hence, CR represents a promising novel conditioning strategy for improving the survival of musculocutaneous flaps with random pattern perfusion.

The Presence of Pre-Existing Endometriotic Lesions Promotes the Growth of New Lesions in the Peritoneal Cavity.

Endometriosis is a common gynecological disease which is characterized by endometriotic lesions outside the uterine cavity. In this study, we investigated whether the presence of pre-existing endometriotic lesions promotes the development of new lesions due to the exchange of cells and an altered peritoneal environment. For this purpose, uterine tissue samples from FVB/N wild-type donor mice were transplanted simultaneously or time-delayed with samples from transgenic FVB-Tg(CAG-luc-GFP)L2G85Chco/J donor mice into the abdominal cavity of FVB/N wild-type recipient mice. The formation of endometriotic lesions was analyzed by means of high-resolution ultrasound, bioluminescence imaging, histology and immunohistochemistry. Moreover, immune cells and inflammatory factors in the peritoneal fluid were assessed by flow cytometry and a cytokine array. These analyses revealed that the growth of newly developing endometriotic lesions is promoted by the presence of pre-existing ones. This is not due to an exchange of cells between both lesion types but rather caused by peritoneal inflammation induced by already established lesions. These findings indicate that, among other pathogenic mechanisms, the chronic nature of endometriosis may be driven by a lesion-induced inflammatory milieu in the peritoneal cavity, which creates favorable conditions for the development of new lesions.

A Photopolymerizable Biocompatible Hyaluronic Acid Hydrogel Promotes Early Articular Cartilage Repair in a Minipig Model In Vivo.

Articular cartilage defects represent an unsolved clinical challenge. Photopolymerizable hydrogels are attractive candidates supporting repair. This study investigates the short-term safety and efficacy of two novel hyaluronic acid (HA)-triethylene glycol (TEG)-coumarin hydrogels photocrosslinked in situ in a clinically relevant large animal model. It is hypothesized that HA-hydrogel-augmented microfracture (MFX) is superior to MFX in enhancing early cartilage repair, and that the molar degree of substitution and concentration of HA affects repair. Chondral full-thickness defects in the knees of adult minipigs are treated with either 1) debridement (No MFX), 2) debridement and MFX, 3) debridement, MFX, and HA hydrogel (30% molar derivatization, 30 mg mL-1 HA; F3) (MFX+F3), and 4) debridement, MFX, and HA hydrogel (40% molar derivatization, 20 mg mL-1 HA; F4) (MFX+F4). After 8 weeks postoperatively, MFX+F3 significantly improves total macroscopic and histological scores compared with all other groups without negative effects, besides significantly enhancing the individual repair parameters "defect architecture," "repair tissue surface" (compared with No MFX, MFX), and "subchondral bone" (compared with MFX). These data indicate that photopolymerizable HA hydrogels enable a favorable metastable microenvironment promoting early chondrogenesis in vivo. This work also uncovers a mechanism for effective HA-augmented cartilage repair by combining lower molar derivatization with higher concentrations.