An accelerated mouse model for atherosclerosis and adipose tissue inflammation.

BACKGROUND: Obesity and particularly the metabolic syndrome, which is often associated with obesity, combine a major risk for type 2 diabetes and cardiovascular disease. Emerging evidence indicate obesity-associated subclinical inflammation primarily originating from adipose tissue as a common cause for type 2 diabetes and cardiovascular disease. However, a suitable and well-characterized mouse model to simultaneously study obesity-associated metabolic disorders and atherosclerosis is not available yet. Here we established and characterized a murine model combining diet-induced obesity and associated adipose tissue inflammation and metabolic deteriorations as well as atherosclerosis, hence reflecting the human situation of cardio-metabolic disease. METHODS: We compared a common high-fat diet with 0.15% cholesterol (HFC), and a high-fat, high-sucrose diet with 0.15% cholesterol (HFSC) fed to LDL receptor-deficient (LDLR-/-) mice. Insulin resistance, glucose tolerance, atherosclerotic lesion formation, hepatic lipid accumulation, and inflammatory gene expression in adipose tissue and liver were assessed. RESULTS: After 12-16 weeks, LDLR-/- mice fed HFSC or HFC developed significant diet-induced obesity, adipose tissue inflammation, insulin resistance, and impaired glucose tolerance compared to lean controls. Notably, HFSC-fed mice developed significantly higher adipose tissue inflammation in parallel with significantly elevated atherosclerotic lesion area compared to those on HFC. Moreover, LDLR-/- mice on HFSC showed increased insulin resistance and impaired glucose tolerance relative to those on HFC. After prolonged feeding (20 weeks), however, no significant differences in inflammatory and metabolic parameters as well as atherosclerotic lesion formation were detectable any more between LDLR-/- mice fed HFSC or HFC. CONCLUSION: The use of high sucrose rather than more complex carbohydrates in high-fat diets significantly accelerates development of obesity-driven metabolic complications and atherosclerotic plaque formation parallel to obesity-induced adipose tissue inflammation in LDLR-/- mice. Hence LDLR-/- mice fed high-fat high-sucrose cholesterol-enriched diet appear to be a suitable and time-saving animal model for cardio-metabolic disease. Moreover our results support the suggested interrelation between adipose tissue inflammation and atherosclerotic plaque formation.

Development of a Dual Reporter System to Simultaneously Visualize Ca2+ Signals and AMPK Activity.

In this study, we introduce a new separation of phases-based activity reporter of kinase (SPARK) for AMP-activated kinase (AMPK), named AMPK-SPARK, which reports the AMPK activation by forming bright fluorescent clusters. Furthermore, we introduce a dual reporter system, named GCaMP-AMPK-SPARK, by incorporating a single-fluorescent protein (FP)-based Ca2+ biosensor, GCaMP6f, into our initial design, enabling simultaneous monitoring of Ca2+ levels and AMPK activity. This system offers the essential quality of information by single-channel fluorescence microscopy without the need for coexpression of different biosensors and elaborate filter layouts to overcome spectral limitations. We used AMPK-SPARK to map endogenous AMPK activity in different cell types and visualized the dynamics of AMPK activation in response to various stimuli. Using GCaMP-AMPK-SPARK, we revealed cell-to-cell heterogeneities in AMPK activation by Ca2+ mobilization. We anticipate that this dual reporter strategy can be employed to study the intricate interplays between different signaling networks and kinase activities.

Does early post-operative exercise influence bone healing kinetics? Preclinical evaluation of non-critical sized femur defect healing.

Physical exercise is a well-known modality for maintaining healthy locomotor mechanism. A detailed preclinical research on physical exercise effect on bone healing kinetics could help to improve the rehabilitation process after fracture treatment and bone remodeling. Our aim was to evaluate the effect of early post-operative exercise effect on bone microstructural changes in a rat model. Twenty Sprague Dawley male rats underwent bi-cortical 1.6 mm hole drilling in both femur diaphysis, after which (n = 10) underwent continuous treadmill training (TR) over two weeks, while the other group of rats (n = 10) was assigned to non-training (NT) control group. New bone formation labeling was performed by subcutaneous fluorochrome injections at day 5, 14 and 31. In vivo micro-computed tomography (µCT) scans were performed once a week during the 6-week post-operative period. Ten animals (five from each group) were euthanized at 3rd week while remaining animals were euthanized at 6th week. Femur samples were extracted and underwent ex vivo µCT and histological evaluation, while serum was used for evaluating alkaline phosphatase (ALP). µCT data demonstrated increased volume and surface of newly formed bone in defect area of TR group. Bone volume/Tissue volume (BV/TV) ratio and number of osteocytes showed an increase in TR group after 3-week period. Fluorochrome distances were increased between day 5 and 14 within the training group. Serum ALP level increased in both groups over 3- and 6-weeks. Post-operative exercise increases the bone healing kinetics and stimulates the new bone formation during and after the training protocol has ended.

The combined effect of zinc and calcium on the biodegradation of ultrahigh-purity magnesium implants.

Magnesium (Mg)-based implants are promising candidates for orthopedic interventions, because of their biocompatibility, good mechanical features, and ability to degrade completely in the body, eliminating the need for an additional removal surgery. In the present study, we synthesized and investigated two Mg-based materials, ultrahigh-purity ZX00 (Mg-Zn-Ca; <0.5 wt% Zn and <0.5 wt% Ca, in wt%; Fe-content <1 ppm) and ultrahigh-purity Mg (XHP-Mg, >99.999 wt% Mg; Fe-content <1 ppm), in vitro and in vivo in juvenile healthy rats to clarify the effect of the alloying elements Zn and Ca on mechanical properties, microstructure, cytocompatibility and degradation rate. Potential differences in bone formation and bone in-growth were also assessed and compared with state-of-the-art non-degradable titanium (Ti)-implanted, sham-operated, and control (non-intervention) groups, using micro-computed tomography, histology and scanning electron microscopy. At 6 and 24 weeks after implantation, serum alkaline phosphatase (ALP), calcium (Ca), and Mg level were measured and bone marrow stromal cells (BMSCs) were isolated for real-time PCR analysis. Results show that ZX00 implants have smaller grain size and superior mechanical properties than XHP-Mg, and that both reveal good biocompatibility in cytocompatibilty tests. ZX00 homogenously degraded with an increased gas accumulation 12 and 24 weeks after implantation, whereas XHP-Mg exhibited higher gas accumulation already at 2 weeks. Serum ALP, Ca, and Mg levels were comparable among all groups and both Mg-based implants led to similar relative expression levels of Alp, Runx2, and Bmp-2 genes at weeks 6 and 24. Histologically, Mg-based implants are superior for new bone tissue formation and bone in-growth compared to Ti implants. Furthermore, by tracking the sequence of multicolor fluorochrome labels, we observed higher mineral apposition rate at week 2 in both Mg-based implants compared to the control groups. Our findings suggest that (i) ZX00 and XHP-Mg support bone formation and remodeling, (ii) both Mg-based implants are superior to Ti implants in terms of new bone tissue formation and osseointegration, and (iii) ZX00 is more favorable due to its lower degradation rate and moderate gas accumulation.

Immunological reaction to magnesium-based implants for orthopedic applications. What do we know so far? A systematic review on in vivo studies.

Magnesium-based implants (Mg) became an attractive candidate in orthopedic surgery due to their valuable properties, such as osteoconductivity, biodegradability, elasticity and mechanical strength. However, previous studies on biodegradable and non-biodegradable metal implants showed that these materials are not inert when placed in vivo as they interact with host defensive mechanisms. The aim of this study was to systematically review available in vivo studies with Mg-based implants that investigated immunological reactions to these implants. The following questions were raised: Do different types of Mg-based implants in terms of shape, size and alloying system cause different extent of immune response? and; Are there missing links to properly understand immunological reactions upon implantation and degradation of Mg-based implants? The database used for the literature research was PubMed (U.S. National Library of Medicine) and it was undertaken in the end of 2021. The inclusion criteria comprised (i) in vivo studies with bony implantation of Mg-based implants and (ii) analysis of the presence of local immune cells or systemic inflammatory parameters. We further excluded any studies involving coated Mg-implants, in vitro studies, and studies in which the implants had no bone contact. The systematic search process was conducted according to PRISMA guidelines. Initially, the search yielded 225 original articles. After reading each article, and based on the inclusion and exclusion criteria, 16 articles were included in the systematic review. In the available studies, Mg-based implants were not found to cause any severe inflammatory reaction, and only a mild to moderate inflammatory potential was attributed to the material. The timeline of foreign body giant cell formation showed to be different between the reviewed studies. The variety of degradation kinetics of different tested implants and discrepancies in studies regarding the time points of immunological investigations impair the conclusion of immunological reactions. This may be induced by different physical properties of an implant such as size, shape and alloying system. Further research is essential to elucidate the underlying mechanisms by which implant degradation affects the immune system. Also, better understanding will facilitate the decision of patients whether to undergo surgery with new device implantation.

Osteotomy after medial malleolus fracture fixed with magnesium screws ZX00 - A case report.

Magnesium alloys have recently become the focus of research, as these implants exhibit suitable biocompatibility and appropriate mechanical properties (Grün et al., 2018 [1]). Through intensive preclinical and clinical investigation, many questions regarding stability, biocompatibility and degradation behavior have been answered (Holweg et al., 2020 [2]). This case report aims to describe handling of these implants in a revision situation, especially when located in situ. To describe available options and relevant considerations, including planning and implementation, a revision surgery of a healed medial malleolus fracture is presented. A medial malleolus fracture was primarily treated by a trauma surgeon with two magnesium screws. Due to an osteochondral lesion of the talus, a revision surgery with osteotomy of the medial malleolus was necessary after 17 months. In this revision, conventional screw removal was not possible due to the degradation of the implant. Taking the degradation and the yield strength of the implant into account, we have chosen on the one hand to over-drill and on the other to leave and perforate the screw. To the best of our knowledge, this is the first case study focusing on the clinical intraoperative site of human bone stabilized with magnesium screws. Despite the hydrogen gas production that occurs during degradation, a solid bone-to-implant interface was evident. With this report, we want to encourage the surgical user to get more involved with resorbable magnesium implants.

Can Hardware Removal be Avoided Using Bioresorbable Mg-Zn-Ca Screws After Medial Malleolar Fracture Fixation? Mid-Term Results of a First-In-Human Study.

Ankle is the most common site of hardware removal, mainly performed within 12 months of the primary surgery. The prominence of the metallic hardware is a frequent cause of pain after fracture fixation. Over the last decade, the development of bioresorbable materials based on magnesium (Mg) has increased. Bioresorbable metals aim to avoid a second surgery for hardware removal. METHODS: Twenty patients with isolated, bimalleolar, or trimalleolar ankle fractures were treated with bioresorbable screws made of Mg, 0.45wt% calcium (Ca) and 0.45wt% zinc (Zn) (ZX00). Patient-reported outcome measures (PROMs) including visual analogue scale (VAS) for pain, the presence of complications 6 and 12 months after surgery and the AOFAS scale after 12 months were reported. The functional outcomes were analysed through the range of motion (ROM) of the ankle joint with a standard goniometer. Degradation products and the bioresorbability of the screws were evaluated using plane radiographs. RESULTS: One patient was lost to follow-up. All patients were free of pain, no complications, shoe conflict or misalignement were reported after 12 months of follow-up. No Mg screws were surgically removed. An additional fixation of the distal fibula or the dorsal tibial fragment with conventional titanium implants (Ti) was performed in 17 patients. Within 12 months after primary refixation, 12 of these patients (71%) underwent a second surgery for Ti hardware removal. The mean AOFAS score was 89.8±7.1 and the difference between the treated and the non-treated site in the ROM of the talocrural joint was 2°±11° after 12 months. Radiolucent areas around the screws were attributed to degradation and did not affect clinical or functional outcomes. After one year, the Mg screw heads could not be detected in the plane radiographs of 17 patients which suggests that the majority of the screw head is degraded without introducing adverse reactions. CONCLUSIONS: At 6 and 12 months, the bioresorbable Mg screws show excellent PROMs without complications or need for screw removal. The resorbability of the screw heads in most of the patients after one year could also provide an advantage over conventional bio-inert implants by avoiding related skin irritation due for instance to shoe conflict.

Implant degradation of low-alloyed Mg-Zn-Ca in osteoporotic, old and juvenile rats.

Implant removal is unnecessary for biodegradable magnesium (Mg)-based implants and, therefore, the related risk for implant-induced fractures is limited. Aging, on the other hand, is associated with low bone-turnover and decreased bone mass and density, and thus increased fracture risk. Osteoporosis is accompanied by Mg deficiency, therefore, we hypothesized that Mg-based implants may support bone formation by Mg2+ ion release in an ovariectomy-induced osteoporotic rat model. Hence, we investigated osseointegration and implant degradation of a low-alloyed, degrading Mg-Zn-Ca implant (ZX00) in ovariectomy-induced osteoporotic (Osteo), old healthy (OH), and juvenile healthy (JH) groups of female Sprague Dawley rats via in vivo micro-computed tomography (µCT). For the Osteo rats, we demonstrate diminished trabecular bone already after 8 weeks upon ovariectomy and significantly enhanced implant volume loss, with correspondingly pronounced gas formation, compared to the OH and JH groups. Sclerotic rim development was observed in about half of the osteoporotic rats, suggesting a prevention from foreign-body and osteonecrosis development. Synchrotron radiation-based µCT confirmed lower bone volume fractions in the Osteo group compared to the OH and JH groups. Qualitative histological analysis additionally visualized the enhanced implant degradation in the Osteo group. To date, ZX00 provides an interesting implant material for young and older healthy patients, but it may not be of advantage in pharmacologically untreated osteoporotic conditions. STATEMENT OF SIGNIFICANCE: Magnesium-based implants are promising candidates for treatment of osteoporotic fractures because of their biodegradable, biomechanical, anti-bacterial and bone regenerative properties. Here we investigate magnesium‒zinc‒calcium implant materials in a rat model with ovariectomy-induced osteoporosis (Osteo group) and compare the related osseointegration and implant degradation with the results obtained for old healthy (OH) and juvenile healthy (JH) rats. The work applied an appropriate disease model for osteoporosis and focused in particular on long-term implant degradation for different bone conditions. Enhanced implant degradation and sclerotic rim formation was observed in osteoporotic rats, which illustrates that the setting of different bone models generates significantly modified clinical outcome. It further illustrated that these differences must be taken into account in future biodegradable implant development.

A lean magnesium-zinc-calcium alloy ZX00 used for bone fracture stabilization in a large growing-animal model.

Over the last decade, demand has increased for developing new, alternative materials in pediatric trauma care to overcome the disadvantages associated with conventional implant materials. Magnesium (Mg)-based alloys seem to adequately fulfill the vision of a homogeneously resorbable, biocompatible, load-bearing and functionally supportive implant. The aim of the present study is to introduce the high-strength, lean alloy Mg‒0.45Zn‒0.45Ca, in wt% (ZX00), and for the first time investigate the clinical applicability of screw osteosynthesis using this alloy that contains no rare-earth elements. The alloy was applied in a growing sheep model with osteotomized bone (simulating a fracture) and compared to a non-osteotomy control group regarding degradation behavior and fracture healing. The alloy exhibits an ultimate tensile strength of 285.7 ± 3.1 MPa, an elongation at fracture of 18.2 ± 2.1%, and a reduced in vitro degradation rate compared to alloys containing higher amounts of Zn. In vivo, no significant difference between the osteotomized bone and the control group was found regarding the change in screw volume over implantation time. Therefore, it can be concluded that the fracture healing process, including its effects on the surrounding area, has no significant influence on degradation behavior. There was also no negative influence from hydrogen-gas formation on fracture healing. Despite the proximal and distal screws showing chronologically different gas release, the osteotomy showed complete consolidation. STATEMENT OF SIGNIFICANCE: Conventional implants involve several disadvantages in pediatric trauma care. Magnesium-based alloys seem to overcome these issues as discussed in the recent literature. This study evaluates the clinical applicability of high-strength lean Mg‒0.45Zn‒0.45Ca (ZX00) screws in a growing-sheep model. Two groups, one including a simulated fracture and one group without fracture, underwent implantation of the alloy and were compared to each other. No significant difference regarding screw volume was observed between the groups. There was no negative influence of hydrogen-gas formation on fracture healing and a complete fracture consolidation was found after 12 weeks for all animals investigated.