Deeksha Malhan

Ex vivo bone culture models preserve aspects of the native three-dimensional bone microenvironment and may provide an intermediate platform for biomaterial screening. However, the application of dynamic-flow whole long-bone defects for time-course evaluation of bone–biomaterial interactions remains insufficiently characterized. We established a dynamic-flow ex vivo rat long-bone defect model and evaluated its capacity to maintain cell viability and to detect biomaterial-associated remodeling responses. Long bones obtained as biological by-products were subjected to standardized metaphyseal defects, with defects either left untreated or filled with injectable bone cement. Explants were maintained under continuous perfusion for 21 days. The presence of viable and membrane-compromised cells at Day 21 was assessed qualitatively by Calcein AM/propidium iodide staining; tissue morphology, mineralized bone and total ossified tissue fractions, and TRAP-positive osteoclast number were assessed on Days 7, 14, and 21. Calcein-positive cells remained detectable in the imaged regions after 21 days of dynamic-flow culture, and mineralized bone was preserved in both groups throughout this period. Cement-treated defects showed a higher mineralized tissue fraction (p < 0.001) and a higher total ossified tissue fraction (p < 0.01) than untreated defects, with no significant effect of culture time on either tissue-fraction endpoint. TRAP-positive osteoclast number was also higher in cement-treated defects (p = 0.004), and the significant Group × Time interaction (p = 0.022) indicates that this difference depended on culture duration. The findings demonstrate the feasibility of using a dynamic-flow whole-bone defect model to study bone–biomaterial interactions ex vivo. The model may serve as an intermediate experimental platform for bone–biomaterial interaction studies before in vivo evaluation.

Various organisms, from single-celled cyanobacteria to more complex multi-cellular life forms including mammals, have evolved an internal timekeeping machinery—the biological clock. The clock generates oscillations in gene and protein expression with a period of ∼24 h, aligning internal time with the Earth’s rotation cycle, and is thus named circadian (circa—about and diem—day). Due to our circadian clock, humans and other animals are able to anticipate, respond, and adapt to environmental changes such as daily light and dark cycles.

Alterations in the circadian system are characteristic of aging on Earth. With the decline in physiological processes due to aging, several health concerns including vision loss, cardiovascular disorders, cognitive impairments, and muscle mass loss arise in elderly populations. Similar health risks are reported as “red flag” risks among astronauts during and after a long‐term Space exploration journey. However, little is known about the common molecular alterations underlying terrestrial aging and space‐related aging in astronauts, and controversial conclusions have been recently reported. In light of the regulatory role of the circadian clock in the maintenance of human health, we review here the overlapping role of the circadian clock both on aging on Earth and spaceflight with a focus on the four most affected systems: visual, cardiovascular, central nervous, and musculoskeletal systems. In this review, we briefly introduce the regulatory role of the circadian clock in specific cellular processes followed by alterations in those processes due to aging. We next summarize the known molecular alterations associated with spaceflight, highlighting involved clock‐regulated genes in space flown Drosophila, nematodes, small mammals, and astronauts. Finally, we discuss common genes that are altered in terms of their expression due to aging on Earth and spaceflight. Altogether, the data elaborated in this review strengthen our hypothesis regarding the timely need to include circadian dysregulation as an emerging hallmark of aging on Earth and beyond.

The circadian clock regulates cellular and molecular processes in mammals across all tissues including skeletal muscle, one of the largest organs in the human body. Dysregulated circadian rhythms are characteristic of aging and crewed spaceflight, associated with, for example, musculoskeletal atrophy. Molecular insights into spaceflight-related alterations of circadian regulation in skeletal muscle are still missing. Here, we investigated potential functional consequences of clock disruptions on skeletal muscle using published omics datasets obtained from spaceflights and other clock-altering, external (fasting and exercise), or internal (aging) conditions on Earth. Our analysis identified alterations of the clock network and skeletal muscle-associated pathways, as a result of spaceflight duration in mice, which resembles aging-related gene expression changes observed in humans on Earth (e.g., ATF4 downregulation, associated with muscle atrophy). Furthermore, according to our results, external factors such as exercise or fasting lead to molecular changes in the core-clock network, which may compensate for the circadian disruption observed during spaceflights. Thus, maintaining circadian functioning is crucial to ameliorate unphysiological alterations and musculoskeletal atrophy reported among astronauts.

The success of fracture healing relies on overlapping but coordinated cellular and molecular events. Characterizing an outline of differential gene regulation throughout successful healing is essential for identifying crucial phase-specific markers and may serve as the basis for engineering these in challenging healing situations. This study analyzed the healing progression of a standard closed femoral fracture model in C57BL/6N (age = 8 weeks) wild-type male mice. The fracture callus was assessed across various days post fracture (D = days 0, 3, 7, 10, 14, 21, and 28) by microarray, with D0 serving as a control. Histological analyses were carried out on samples from D7 until D28 to support the molecular findings. Microarray analysis revealed a differential regulation of immune response, angiogenesis, ossification, extracellular matrix regulation, mitochondrial and ribosomal genes during healing. In-depth analysis showed differential regulation of mitochondrial and ribosomal genes during the initial phase of healing. Furthermore, the differential gene expression showed an essential role of Serpin Family F Member 1 over the well-known Vascular Endothelial Growth Factor in angiogenesis, especially during the inflammatory phase. The significant upregulation of matrix metalloproteinase 13 and bone sialoprotein from D3 until D21 asserts their importance in bone mineralization. The study also shows type I collagen around osteocytes located in the ossified region at the periosteal surface during the first week of healing. Histological analysis of matrix extracellular phosphoglycoprotein and extracellular signal-regulated kinase stressed their roles in bone homeostasis and the physiological bone-healing process. This study reveals previously unknown and novel candidates, that could serve as a target for specific time points in healing and to remedy cases of impaired healing.

The circadian clock coordinates the timing of several cellular processes including transcription, the cell cycle, and metabolism. Disruptions in the clock machinery trigger the abnormal regulation of cancer hallmarks, impair cellular homeostasis, and stimulate tumourigenesis. Here we investigated the role of a disrupted clock by knocking out or knocking down the core-clock (CC) genes ARNTL, PER2 or NR1D1 in cancer progression (e.g., cell proliferation and invasion) using colorectal cancer (CRC) cell lines HCT116, SW480 and SW620, from different progression stages with distinct clock phenotypes, and identified mechanistic links from the clock to altered cancer-promoting cellular properties. We identified MACC1 (metastasis-associated in colon cancer 1), a known driver for metastasis and an EMT (epithelial-to-mesenchymal transition)-related gene, to be significantly differentially expressed in CC manipulated cells and analysed the effect of MACC1 manipulation (knockout or overexpression) in terms of circadian clock phenotype as well as cancer progression. Our data points to a bi-directional MACC1-circadian clock interplay in CRC, via CC genes. In particular, knocking out MACC1 reduced the period of oscillations, while its overexpression increased it. Interestingly, we found the MACC1 protein to be circadian expressed in HCT116 WT cells, which was disrupted after the knockout of CC genes, and identified a MACC1-NR1D1 protein–protein interaction. In addition, MACC1 manipulation and CC knockout altered cell invasion properties of HCT116 cells, pointing to a regulation of clock and cancer progression in CRC, possibly via the interaction of MACC1 with core-clock genes.

Emerging evidence points towards a regulatory role of the circadian clock in alternative splicing (AS). Whether alterations in core-clock components may contribute to differential AS events is largely unknown. To address this, we carried out a computational analysis on recently generated time-series RNA-seq datasets from three core-clock knockout (KO) genes (ARNTL, NR1D1, PER2) and WT of a colorectal cancer (CRC) cell line, and time-series RNA-seq datasets for additional CRC and Hodgkin’s lymphoma (HL) cells, murine WT, Arntl KO, and Nr1d1/2 KO, and murine SCN WT tissue. The deletion of individual core-clock genes resulted in the loss of circadian expression in crucial spliceosome components such as SF3A1 (in ARNTL^KO), SNW1 (in NR1D1^KO), and HNRNPC (in PER2^KO), which led to a differential pattern of KO-specific AS events. All HCT116^KO cells showed a rhythmicity loss of a crucial spliceosome gene U2AF1, which was also not rhythmic in higher progression stage CRC and HL cancer cells. AS analysis revealed an increase in alternative first exon events specific to PER2 and NR1D1 KO in HCT116 cells, and a KO-specific change in expression and rhythmicity pattern of AS transcripts related to cancer hallmarks genes including FGFR2 in HCT116_ARNTL^KO, CD44 in HCT116_NR1D1^KO, and MET in HCT116_PER2^KO. KO-specific changes in rhythmic properties of known spliced variants of these genes (e.g. FGFR2 IIIb/FGFR2 IIIc) correlated with epithelial-mesenchymal-transition signalling. Altogether, our bioinformatic analysis highlights a role for the circadian clock in the regulation of AS, and reveals a potential impact of clock disruption in aberrant splicing in cancer hallmark genes.

Osseointegration is a prerequisite for the long-term success of implants. Titanium implants are preferred for their biocompatibility and mechanical properties. Nonetheless, the need for early and immediate loading requires enhancing these properties by adding bioactive coatings. In this preclinical study, extracellular matrix properties and cellular balance at the implant/bone interface was examined. Polyelectrolyte multilayers of chitosan and gelatin or with chitosan and Hyaluronic acid fabricated on titanium alloy using a layer-by-layer self-assembly process were compared with native titanium alloy. The study aimed to histologically evaluate bone parameters that correlate to the biomechanical anchorage enhancement resulted from bioactive coatings of titanium implants in a rat animal model. Superior collagen fiber arrangements and an increased number of active osteocytes reflected a significant improvement of bone matrix quality at the bone interface of the chitosan/gelatin-coated titan implants over chitosan/hyaluronic acid-coated and native implants. Furthermore, the numbers and localization of osteoblasts and osteoclasts in the reparative and remodeling phases suggested a better cellular balance in the chitosan/Gel-coated group over the other two groups. Investigating the micro-mechanical properties of bone tissue at the interface can elucidate detailed discrepancies between different promising bioactive coatings of titanium alloys to maximize their benefit in future medical applications.

Background Merino land sheep are a popular pre-clinical large animal model in research on systemic skeletal diseases such as osteoporosis. Interpretation of studies is difficult because many reference parameters are missing or not established. This study aims to determine the reference parameters of the skeletal system (peak bone mass = PBM, T-Score). A defined standard allows an easier comparison of the study data of the animal model with human studies (T-Score). Materials and methods A total of 116 Dual Energy X-ray Absorptiometry DXA measurements were performed on 74 untreated sheep. The average age of the animals was 57 months. The BMD, BMC, and fat content of the sheep were determined by the relevant human region of interest (ROI). From this, the PBM and from this the T-score for each of the animals were calculated. Results Using 682 DXA measurements BMD and BMC were determined to provide an indication to PBM. For BMD a significant correlation to the age of the animals was observed (p = 0.043). A significant correlation was also seen for BMC (B) (p ≤ 0.001). In the age-dependent analysis, a widespread of values above the linear regression line was measured for both BMD and BMC between the 50th and 90th months of life. From an age of about 90 months, a wider spread of values below the linear regression line was found, although the average values continued to rise. Discussion The evaluation of the 116 DXA measurements allowed the determination of the PBM for merino land sheep. With the help of the PBM, a T-score was calculated for each animal. The statistical analysis shows significant differences in BMD values between the different animal groups in each of the four ROIs investigated. Individual control or sham groups per study are therefore not sufficient. To improve comparability, an independent reference group should be established. Conclusion An independent reference group for PBM and a T-score was established from four to six-year-old animals. The bone density increases with the age of the animals. Around the fourth year of life, a first peak could be observed. Also, after the seventh year of life, a further peak with the beginning plateau phase was observed. When compiling a group of animals for an osteoporosis model, animals from the age of seven years should, therefore, be used.

Hypodontia often leads to limited bone availability of the alveolar ridges. Oral rehabilitation of severe hypodontia patients is challenging. In this retrospective study, we evaluated the functional and aesthetic results after dental implants in hypodontia patients, corroborated by Albrektsson implant success criteria. Over a period of 15 years (2000–2015), a total of 43 patients were diagnosed with hypodontia and 165 dental implants were inserted. Six patients who received 10 implants were lost in the follow-up. We examined 155 implants in 37 patients between December 2015 and May 2017. Besides family history, patients evaluated the general satisfaction, functionality, and aesthetics of the implants. Study subjects were between 17 and 44 years old (mean ± SD: 21.4 ± 5.6). Hypodontia patients were missing one to five teeth (n = 28), whereas patients diagnosed with oligodontia (≥6 missing teeth, n = 9). In this study, 24 patients (64.9%) with hypodontia had a positive family history; the remaining 13 patients had no family member with hypodontia. The final follow-up time ranged between 5 and 189 months after implant placement. Orthodontic treatment was performed in 32 patients (86%) before implant placement. Rehabilitation resulted in 62% of the cases being treated with 1–2 implants and 38% treated with 3–15 implants. However, out of 155 inserted dental implants, 18 implants failed to meet Albrektsson criteria, under which two implants were removed. Only autografts were used for bone augmentation with 97 implants. More than two-thirds of the patients showed high general satisfaction and masticatory function (69.4%) as well as phonetic ability (80.6%). The aesthetic outcome was rated as excellent by 17 patients (47.2%). The findings emphasize the importance of interdisciplinary treatment of hypodontia, leading to a satisfactory, functional, and long-term fixed prosthodontics using dental implants.