Young Bone Research Articles

Efficacy of Bone Marrow Derived MSC for Steroid Refractory Acute GVHD Associates with Age and a Defined Molecular Profile of MSC Donors

IntroductionAcute graft versus host disease (aGVHD) remains a life-threatening complication and substantially reduces efficacy of allo-HSCT. In particular, the outcome of patients with severe steroid-refractory aGVHD continues to be poor. Administration of Mesenchymal Stromal Cells (MSC) has been reported by others and us as an interesting treatment option (Le, Frassoni et al. 2008, von, Stolzel et al. 2009, Lucchini, Introna et al. 2010, Te Boome, Mansilla et al. 2015). Though insurance and fast track approval has recently been granted to MSC products in Japan and USA, variability arising from donor variability and the inability to link surrogate markers of the product to clinical efficacy could prove to be a major obstacle in daily clinical practice.Materials and MethodsWe evaluated the impact of individual MSC donors as well as MSC donor properties such as age on clinical response in a cohort of 102 patients with grade II-IV steroid refractory aGVHD treated within a recently reported prospective study as well as an extended hospital exemption program (Te Boome, Mansilla et al. 2015). Bone marrow derived MSC from third party non-HLA matched donors were administered at day 1, 8, and 21 as reported (Te Boome, Mansilla et al. 2015). Primary outcome measures were one year overall survival (OS) and response of GVHD. Cox proportional hazards models, competing risk analyses (Gray's test) and Kaplan Meier estimates were used for analyzing response and OS respectively. Two different R statistical packages for RNA-seq analysis were used for the calculation of differentially expressed genes (DEGs) (DeSeq & EdgeR). The DEGs were considered significant based on the overlap across the two methods and a False Discovery Rate/adjusted p-value<0.05.Results102 patients received in total 299 MSC infusions derived from 10 different BM donors. Median number of infusions was 3 (range 1-4). 75,5% of patients received all MSC infusions from the same donor, 20,6% with MSC from 2 donors and 3,9% with MSC from 3 different donors. Two donors were used to treat 28,4% and 43,1% of patients respectively. When testing retrospectively impact on 1 year OS of an individual product no differences between patients treated with either the 2 main contributing donors or the patients treated with the ‘other’ MSC donors could be observed. However, when donor age was used to cluster cohorts (donor age < and > 10 years) differences were observed (Figure 1) with a survival benefit for patients treated with young donors. Competing risk analysis also revealed a significant benefit for patients only treated with young donors. Furthermore, in multivariate analysis, MSC donor age remained predictive for OS (HR 2,00, p-value 0,025). RNA sequencing analysis was used to further dissect molecular differences between individual donors and allowed to identify a distinctive molecular profile active in MSC that associates with improved clinical outcome.ConclusionsIn our cohort of 102 patients with steroid refractory acute GVHD that were treated with MSC we observed in this retrospective study differences in achieving both complete resolution of GVHD symptoms and 1 year OS. Patients only treated with MSC derived from very young bone marrow donors (<10 years old) had significantly better resolution of GVHD symptoms as well as an improved OS. Furthermore we identified a distinctive molecular profile by means of RNA sequencing that associates with improved clinical outcome. These findings may have implications for current products entering the market approval as well as on both future as well as currently ongoing clinical trials with MSC. [Display omitted] DisclosuresKuball:Miltenyi: Research Funding; Gadeta (www.gadeta.nl): Consultancy, Equity Ownership, Patents & Royalties: on gdT cells and receptors and isolation strategies , Research Funding.

Calcaneal Ultrasound and Its Relation to Dietary and Lifestyle Factors, Anthropometry, and Vitamin D Deficiency in Young Medical Students.

BackgroundBone quality and peak bone mass are greatly affected by lifestyle factors. The objective of the study was to investigate the relationships between anthropometry, dietary and caloric intake, body composition measurements, physical activity, and vitamin D status with quantitative ultrasound-based bone parameters among medical students.MethodsBoth male and female medical college students were included in this study. A detailed questionnaire was administered, collecting clinical, dietary, physical activity information, physical examination details, including body mass index (BMI). Body composition (total body fat, total body water, muscle mass, mean visceral fat mass, basal metabolic rate, bone mass using a bioelectrical impedance analyzer) and calcaneal heel ultrasound parameters were measured using an Osteosys Sonost-3000, Ultrasound Bone Densitometer were measured, respectively.ResultsIn this cross-sectional study, 211 healthy students with a mean age of 20.1 ± 1.1 years, 51.7% (n = 109) were males. Majority (79.4%) of the young adults had vitamin D deficiency. The mean BMI, calcium intake, and vitamin D levels were 22.35 ± 3.43 kg/m2, 788.7 ± 364.8 mg/day, and 15.02 ± 8.63 ng/ml, respectively. Female subjects compared to males had statistically significantly lower daily energy intake, muscle mass, visceral fat mass, calcium intake, and vitamin D levels. In addition the median Z-scores in females [−1.40 (−0.57 to −1.82)] was significantly poorer than the male [−0.50 (0.20 to −1.3)] counterparts, p-value <0.001. Multiple regression analysis showed that overall body fat percent (p-value 0.016) and visceral fat percent (p-value 0.029) were the only significant negative predictors to the calcaneal bone quality index (BQI) values.ConclusionAdolescent lifestyle patterns can influence young adult bone strength. The young Pakistani females exhibited significantly lower dietary intakes and more inadequate bone parameters compared to males. Our data suggest that total body and visceral fat percent are the predominant negatively associated determinant of bone strength for this cohort. Calcaneal ultrasound can be utilized for mass screening of young adults for identification of low BMD.

Prioritization of Genes Relevant to Bone Fragility Through the Unbiased Integration of Aging Mouse Bone Transcriptomics and Human GWAS Analyses.

Identifying new genetic determinants of bone mineral density (BMD) and fracture promises to yield improved diagnostics and therapies for bone fragility. However, prioritizing candidate genes from genome-wide screens can be challenging. To overcome this challenge, we prioritized mouse genes that are differentially expressed in aging mouse bone based on whether their human homolog is associated with human BMD and/or fracture. Unbiased RNA-seq analysis of young and old male C57BL/6 mouse cortical bone identified 1499, 1685, and 5525 differentially expressed genes (DEGs) in 1, 2, and 2.5-year-old bone, relative to 2-month-old bone, respectively. Gene-based scores for heel ultrasound bone mineral density (eBMD) and fracture were estimated using published genome-wide association studies (GWAS) results of these traits in the UK Biobank. Enrichment analysis showed that mouse bone DEG sets for all three age groups, relative to young bone, are significantly enriched for eBMD, but only the oldest two DEG sets are enriched for fracture. Using gene-based scores, this approach prioritizes among thousands of DEGs by a factor of 5- to 100-fold, yielding 10 and 21 genes significantly associated with fracture in the two oldest groups of mouse DEGs. Though these genes were not the most differentially expressed, they included Sost, Lrp5, and others with well-established functions in bone. Several others have, as yet, unknown roles in the skeleton. Therefore, this study accelerates identification of new genetic determinants of bone fragility by prioritizing a clinically relevant and experimentally tractable number of candidate genes for functional analysis. Finally, we provide a website (www.mouse2human.org) to enable other researchers to easily apply our strategy. © 2022 American Society for Bone and Mineral Research (ASBMR).

Limited rejuvenation of aged hematopoietic stem cells in young bone marrow niche.

Hematopoietic stem cells (HSCs) exhibit functional alterations, such as reduced regenerative capacity and myeloid-biased differentiation, with age. The HSC niche, which is essential for the maintenance of HSCs, also undergoes marked changes with aging. However, it has been technically challenging to directly evaluate the contribution of niche aging to age-associated HSC alterations without niche-damaging myeloablation in HSC transplantation assays. We herein transplanted an excess of aged HSCs into young mice without preconditioning. Although aged HSCs successfully engrafted in the intact young bone marrow niche, they poorly regenerated downstream progenitors and exhibited persistent myeloid-biased differentiation, resulting in no significant functional rejuvenation. Transcriptome and methylome analyses revealed that the young niche largely restored the transcriptional profile of aged HSCs, but not their DNA methylation profiles. Therefore, the restoration of the young niche is insufficient for rejuvenating HSC functions, highlighting a key role for age-associated cell-intrinsic defects in HSC aging.

Redox Potential and Antioxidant Capacity of Bovine Bone Collagen Peptides towards Stable Free Radicals, and Bovine Meat Lipids and Proteins. Effect of Animal Age, Bone Anatomy and Proteases-A Step Forward towards Collagen-Rich Tissue Valorisation.

Collagen antioxidant peptides are being widely studied. However, no research has paid attention to biological parameters such as the age and anatomy of collagen-rich tissues, which can determine a change in tissue structure and composition, and then in bioactivity. Moreover, only few research works have studied and assessed peptides antioxidant activity on the food matrix. This work aimed to investigate the effect of bovine’s bone age and anatomy, and of six different enzymes, on the antioxidant activity of collagen peptides. Collagen was extracted from young and old bovine femur and tibia; six different enzymes were used for peptides’ release. The redox potential, the quenching of stable free radicals, and the antioxidant capacity on bovine meat lipids and proteins was evaluated, under heating from ambient temperature to 80 °C. Age and anatomy showed a significant effect; the influence of anatomy becomes most important with age. Each enzyme’s effectiveness toward age and anatomy was not the same. The greatest amount of peptides was released from young bones’ collagen hydrolysed with papain. The antioxidant activity was higher at higher temperatures, except for meat proteins. Assessing the effect of age and anatomy of collagen-rich tissues can promote a better application of collagen bioactive peptides.

Raman spectroscopy reveals age- and sex-related differences in cortical bone from people with osteoarthritis

Bone strength in human cortical bone is determined by the composition and structure of both the mineral and collagen matrices and influenced by factors such as age, gender, health, lifestyle and genetic factors. Age-related changes in the bone matrix are known to result in loss of mechanical strength and increased fragility. In this study we show how Raman spectroscopy, with its exquisite sensitivity to the molecular structure of bone, reveals new insights into age- and sex-related differences. Raman analysis of 18 samples of cortical hip bone obtained from people aged between 47–82 years with osteoarthritis (OA) found subtle changes in the lipid and collagen secondary structure, and the carbonate (CO32−) and phosphate (PO43−) mineral ratios in the bone matrix. Significant differences were observed between older and younger bones, and between older female and older male bones; no significant differences were observed between younger male and female bones. Older female bones presented the lowest mineral to matrix ratios (MMR) and highest CO32−/PO43− ratios, and relative to lipid/collagen –CH2 deformation modes at 1450 cm−1 they had lowest overall mineral content, higher collagen cross linking and lipid content but lower levels of α-helix collagen structures than older male and younger male and female bones. These observations provided further insight on bone composition changes observed in the bone volume fraction (BV/TV) for the older female bones from microCT measurements on the same samples, while tissue mineral density (TMD) measurements had shown no significant differences between the samples.

Age-Associated Changes in Bone Marrow-Derived Extracellular Vesicles May Alter Their Effects on Murine Hematopoietic Stem Cell Function

Extracellular vesicles (EVs) are critical mediators of intercellular communication within the bone marrow niche and have been implicated in numerous features of aging. However, their role in natural hematopoietic stem cell (HSC) aging has not been fully elucidated. The goal of this work was to test the hypothesis that EVs from whole bone marrow (BM-EVs) can modulate the HSC aging phenotype in vivo. With respect to HSC aging, our prior work showed that, in contrast to the well-known reduced functional capacity and prominent myeloid skewing displayed by old immunophenotypically-defined HSCs, old unseparated whole bone marrow (WBM) (24-26-mo-old) had a 4-fold increase in functional HSCs compared to young (6-8-wk-old) WBM in limit dilution competitive bone marrow transplantation, and showed minimal to no myeloid skewing. In order to test the ability of BM-EVs derived from this total WBM population to alter HSC aging phenotype, we first isolated EVs from WBM flushed from old (24-26-month old) and young (6-8-week old) C57/BL6 (CD45.2) mice by differential centrifugation (2000 × g for 30 min, supernatant centrifuged 100,000 × g for 1 hour, BM-EV pellet collected). Utilizing nanoparticle tracking analysis, we found no difference in mean particle size between old and young BM-EVs, but there was an approximately 2-fold increase in the number of EVs from old WBM compared to young WBM. To test the ability of these BM-EVs to alter HSC function in vivo, we injected old CD45.2 mice with 2 x 109 young BM-EVs and young CD45.2 mice with 2 x 109 old BM-EVs mice via tail vein, daily x 3 days. Control mice were injected with age-matched BM-EVs or vehicle alone. At one-month post injection, we isolated total WBM and Lineage negative/c-Kit+/Sca-1+/CD150+ cells (LSK-SLAM) from the EV-exposed or vehicle control mice. We injected either 3 x 105 WBM cells or 400 LSK-SLAM mixed with 3 x 105 healthy WBM competitor cells from young B6.SJL (CD45.1) mice into lethally irradiated young CD45.1 hosts and measured peripheral blood chimerism and lineage contribution by flow cytometry up to 6 months post-transplant. For the young marrow, exposure to old BM-EVs had no appreciable effects on engraftment capacity or lineage distribution. However, old WBM exposed to young BM-EVs exhibited a significant decrease in engraftment (15% ± 5%) when compared to old WBM exposed to age-matched old BM-EVs (61% ± 14%) or vehicle control (47% ± 7%) (% average donor chimerism ± SEM, n=4-5 mice/group, p&amp;lt;0.04). Similarly, there was a trend toward decreased engraftment capacity by old LSK-SLAM after young BM-EV exposure (7% ± 4%) and increased engraftment capacity by the old LSK-SLAM after old BM-EV exposure (27% ± 10%) compared to vehicle control (15% ± 2%) (% average donor chimerism ± SEM, n=4-5 mice/group, p=not significant (ns)). These results are consistent with our prior data discussed above in which old un-separated WBM, the source of the old BM-EVs, had increased engraftment capacity compared to young WBM. Interestingly, there was also a trend toward reversal of classic myeloid skewing when old LSK-SLAM were exposed to young BM-EVs (36% ± 11%) compared to vehicle control (64% ± 3%) (average myeloid % of donor-derived peripheral blood ± SEM, n=4-5mice/group, p=ns). In addition, consistent with the known increase in LSK-SLAM with age, our preliminary data showed that old mice exposed to young BM-EVs had an approximately 7-fold decrease in the number of LSK-SLAM in marrow, indicating that BM-EVs within the young bone marrow microenvironment may modulate HSC population size. Finally, microRNA (miR) expression profiling (NanoString Technologies) indicated that a number of miRs known to be involved in hematopoiesis, proliferation, self-renewal, differentiation, senescence and inflammation were differentially represented in old and young BM-EVs, with miR-29a, miR-24, and miR-21 significantly increased and miR-105 significantly decreased in old BM-EVs compared to young BM-EVs. Together, these data indicate that young BM-EVs, via transfer of differentially age-related cargo, may alter engraftment capacity, modify the lineage commitment and may regulate LSK-SLAM population size during natural aging. Future studies more clearly delineating age-associated BM-EV effects on HSCs and defining the molecular mechanisms underlying these effects could yield key insights into the natural aging of HSCs and facilitate restoration of healthy hematopoiesis in the elderly. Disclosures No relevant conflicts of interest to declare.

Deproteinized young bone reveals a continuous mineral phase and its contribution to mechanical properties with age

Bone is composed of mineral (apatite crystals) phase, organic (proteins, mainly collagen, and other organics) phase, and water at the nanoscale. Mineral contributes to elastic modulus and strength while organics and water give bone its toughness. Changes in the mineral composition and arrangement with age during development, and the resulting mechanical properties, are of high scientific and clinical interests. The mineral phase can be studied by removing the organic phase from bone using sodium hypochlorite (NaClO). This process is called deproteinization (DP). In this paper, four age groups (3-week, 4-week, 16-week, and 24-week) were compared in both untreated and DP bone states. We find that the deproteinized bone samples from all these age groups are self-standing. Thus, the mineral phase forms a continuous structure, even in the 3-week bone. Micro-computed tomography imaging displays a significant porosity decrease with the increase in age. Raman spectroscopy reveals increases in both the carbonate-to-phosphate ratio and the mineral crystallinity with age. Compression test results show that the elastic modulus and strength of the mineral phase alone are positively related to age. The elastic modulus of DP bone is less affected by age than that of the untreated bone. Overall, the mechanical properties of the mineral phase increase with age due to a decrease in porosity and an increase in the mineral content.

The Effect of Resection Size in the Treatment of Cam-Type Femoroacetabular Impingement in the Typical Patient With Hip Arthroscopy: A Biomechanical Analysis

Background: Arthroscopic osteochondroplasty may improve range of motion and relieve pain in patients with symptomatic hip impingement. Femoral neck fracture is a risk of this procedure because of the weakening of the proximal femur. To our knowledge, there are no biomechanical studies in young human cadaveric bone evaluating the effect of osteochondroplasty on femoral neck strength. Purpose/Hypothesis: The purpose was to evaluate loads to fracture in young human cadavers after resection depths of 25% and 40% at the head-neck junction. We hypothesized that both depths will maintain ultimate loads to failure above previously published loads, as well as above physiologic weightbearing loads. Study Design: Descriptive laboratory study. Methods: Cadaveric proximal femoral specimens (6 matched pairs, under the age of 47 years) were divided into 2 groups: 25% or 40% of the diameter at the head-neck junction was resected. The length of the resection was 2 cm and the width of the resection was determined by the length of the anterolateral quadrant at the head-neck junction in all cases. A compressive load was applied directly to the femoral head. Peak load, stiffness, and energy to fracture were compared between groups. Results: The average peak load to fracture after 25% resection (7347 N) was significantly higher than after the 40% resection (5892 N) (P = .010). The average energy to fracture was also significantly higher in the 25% resection group (30.2 J vs 19.2 J; P = .007). The average stiffness was higher in the 25% group, although not statistically significant (P = .737). Conclusion: Resection depths of 25% and 40% at the anterolateral quadrant of the femoral head-neck junction may be safe at previously described functional loads such as standing and walking in the age range more typically seen in patients undergoing hip arthroscopy. Loads to fracture were significantly higher than previously reported using older cadaveric specimens. Clinical Relevance: Currently, most surgeons limit weightbearing after femoral osteochondroplasty in part because of risk of femoral neck fracture. Given the higher observed loads to fracture, young patients could possibly bear weight sooner after surgery, although postoperative protocols should be individualized based on patient age, weight, bone density, amount of bone resected, concomitant procedures, and potential compliance with activity restrictions.

Abstract 1717: Targeted NanoSpheres (TNS): An effective, non-toxic nano-formulated therapeutic platform for broad use in cancer treatment

Abstract Current cancer therapy relies on the use of cytotoxic agents (‘chemotherapy') that was developed over 50 years ago. These agents are very effective, but efficacy is limited by off-target toxicity to normal tissues. It is widely recognized that chemotherapy could be far more effective if higher doses could be delivered to the tumor and not to normal tissues. By refining the early formulations and after extensive testing, we developed a novel targeted nanoparticle called Targeted NanoSpheres or TNS. We showed this novel nano-formulated platform can overcome many deficiencies of conventional nanoparticles and therefore has enhanced potential as an effective delivery vehicle for virtually any type of cancer for which a suitable tumor cell surface antigen is present. We are exploring the development of NV103, a first-in-class anti-CD99 TNS (CD99-TNS/Ir), for the treatment of Ewing Sarcoma (EWS), a highly lethal pediatric and young adult bone and soft tissue tumor driven by a fusion protein, typically EWS/FLI1 in over 85% of cases. This platform is also being investigated for other types of cancer that either express CD99, or other tumor antigens like GD2 and B7-H3. In addition, a variety of payloads, including small molecules chemotherapeutics like irinotecan and doxorubicin, as well as plasmids, siRNA, ASOs, and mRNA, have been successfully incorporated and effectively delivered with documented superior anti-tumor effect. NV103 itself showed significant anti-tumor effect in hepatocellular carcinoma and prostate tumor cell lines and xenografts. In all cases, systemic toxicity is minimized by use of targeting antibodies that enhance tumor delivery while minimizing systemic exposure of normal tissues. Citation Format: Hyunggyoo Kang, Jon Nagy, Timothy Triche. Targeted NanoSpheres (TNS): An effective, non-toxic nano-formulated therapeutic platform for broad use in cancer treatment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1717.

Effect of Aging and 5-Fluorouracil Treatment on Bone Marrow Stem Cell Dynamics.

Lifelong homeostasis of bone marrow is maintained by the resident stem cells that include the quiescent very small embryonic-like stem cells (VSELs) and lineage restricted, tissue committed 'progenitors' hematopoietic stem cells (HSCs). Niche providing mesenchymal stromal cells (MSCs) regulate the function of VSELs/HSCs by providing crucial paracrine support. Any dysfunction of stem cells and/or their niche leads to disease state. The stem cells biology gets affected with age leading to a myeloid bias in differentiation of HSCs and increased incidence of myeloid leukemia. Present study was undertaken to enumerate VSELs, HSCs and MSCs and evaluate their response on D4 and D10 after chemotherapy with 5-Fluorouracil (5-FU) in young and aged mouse bone marrow. Stem cells were activated in response to 5-FU induced stress in an attempt to restore homeostasis. Although absolute numbers of VSELs and HSCs did not differ much between young and aged mice, their tendency to proliferate was higher on D4 in aged mice. However, ability to revert back to basal numbers and their differentiation was affected on D10 in aged marrow. Stem cells from aged bone marrow showed greater ability to form CFUs on D10 after 5-FU treatment. CD44 positive aged MSCs also showed increased proliferation on D10. Transplanting MSCs from young mice in 5-FU treated aged marrow helped improve hematopoiesis. The results suggest thatno significant intrinsic changes occur asproliferative ability of stem cells remains unaffectedbut the niche gets affected with age leading to excessive self-renewal and compromised differentiation. This may explain increased incidence of leukemia with age.

Pilot in vivo animal study of bone regeneration by Laser Patterned Microcoagulation technology

Relevance. Fractional laser technologies are very popular. The ability to create microchannels (coagulation or ablative nature) in the tissue by laser radiation stimulating its regeneration is the basis of fractional technology. The histological structure of the rabbit parietal bone during its regeneration after Laser Patterned Microcoagulation treatment was investigated by hematoxylin and eosin (H&amp;E) stain.Purpose. This in vivo study investigated the regeneration of the rabbit’s parietal bone after fractional laser treatment using diode lasers with wavelengths 980 nm and 1550 nm.Materials and methods. A study was performed on the bone tissue of 10 laboratory animals (rabbits) using laser fractional treatment with wavelengths 1.55 and 0.98 μm. As laboratory animals (rabbits) were used Soviet chinchillas weighing 2.5-3 kg. 2 different lasers were used: FONALaser (Sirona Dental Systems, Germany) with a wavelength of 980 nm, a power of 5 W and a laser surgical device (LSP “IRE-Polyus”, Russia) with a wavelength of 1550 nm, a power of 25 W and a pulse width range from 60 to 250 ms.Results. On day 21, the thickness of the periosteum in the group with a wavelength of 980 nm did not significantly differ from the thickness of the periosteum in the group with a wavelength of 1550 nm – p = 0.4000. On the 45th day of observation, there were also no significant differences in the thickness of the periosteum between both groups – p = 0.2000.Conclusion. It was shown that the most destruction processes were noted by laser with a wavelength 1550 nm in the experiment without periosteum. The smallest changes were noted by laser with a wavelength 980 nm in the presence of periosteum. In this study, in 6 cases out of 10, the formation of young bone tissue as thin strip form at the bottom of the lesion site was noted.

Neuropeptide Y1 receptor antagonist but not neuropeptide Y itself increased bone mineral density when locally injected with hyaluronic acid in male Wistar rats

Background/aim The nervous system controls bone mass via both the central (CNS) and the peripheral (PNS) nervous systems. Intriguingly, neuropeptide Y (NPY) signaling occurs in both. Less is known on how the PNS stimulated NPY signaling controls bone metabolism. The objective of this study was to evaluate whether NPY or NPY1 receptor antagonist changes local bone mineral density (BMD) when injected into a Wistar rat tibia. Materials and methodsTibial intramedullary area of 24 wild type male Wistar rats (average weight = 350 ± 50 g, average age = 4 ± 0.5 months) were injected with NPY (1 × 10-5 M and 1 × 10-6 M) and NPY1 receptor antagonist (1 × 10-4 M) dissolved in hyaluronic acid (HA) separately. Tibiae were collected after one and two weeks. BMD was measured with dual-energy X-ray absorptiometry (DXA) and micro quantitative computer tomography (QCT). Histological changes were analyzed with light microscopy, Goldner's Masson trichrome (MT), and hematoxylin-eosin staining.ResultsAccording to DXA , the mean BMD of NPY dose 1 (1 × 10-5 M) was significantly lower than that of the control (HA applied) group and not significantly but still lower than that of the NPY dose 2 and NPY1 antagonist applied groups. QCT results indicated the same pattern statistically insignificantly in the trabecular area but not in the cortex of the bones. Histologically, only NPY1 antagonist applied tibiae revealed young spongiosis bone trabeculae formed in the borderline of the cortical bones. HA was remarkably biocompatible and late degrading in the tissues. Conclusion Local administration of NPY and NPY1 antagonists may hold regulating potential of BMD and bone formation. NPY1 antagonist caused new bone formation in trabecular bone when applied locally. NPY dissolved in HA however can be used to suppress bone formation.

Effect of temperature on functional activity of macrophages in three different species

Abstract Temperature affects body physiological functions, including immunity in ways that influence survival. This study investigated the effect of temperature on macrophage activation and metabolism across species. Macrophages were isolated from mice, chicken or rainbow trout primary tissue or from macrophage cell lines and were activated with lipopolysaccharide (LPS) or IFN-γ. For mice, both Raw264.7 and primary cells, nitric oxide (NO) production was similar at 35°C and 37°C but dropped dramatically at temperatures below 35°C. At fever temperature (39°C), NO release increased in response to LPS. Young bone marrow derived macrophage (BMDM) and peritoneal resident macrophage (PRM) showed increased protein synthesis at 39°C compared to 37°C. Chicken splenic macrophages (CSM) showed NO responses that were similar at 37, 39, and at 41°C (normal for avian). A fever of 42°C had a large stimulatory effect on NO production compared to 41°C. A chicken liver derived macrophage cell line, HTC, showed the same pattern. They also showed higher protein synthesis at 42°C compared with (41°C) after LPS stimulation. Trout head-kidney macrophages (THM) showed the highest NO responses at 19°C when compared to more typical stream temperatures of 13, 15, and 16°C. They still showed some response at 28 and 37°C. Thus, their macrophages respond at higher temperatures than the fish can tolerate. Stimulated THM cells with LPS at 19°C showed increased protein synthesis compared to 13 and 16°C. We can conclude from our experiments that fish macrophages had a much broader range of temperatures at which they could respond by NO generation and protein synthesis compared to mice and chickens and all had increased responses at fever temperatures.

Effects of age on retinal macrophage responses to acute elevation of intraocular pressure

There is accumulating evidence that aging shifts the central nervous system milieu towards a proinflammatory state, with increased reactivity of microglia in the aging eye and brain having been implicated in the development of age-related neurodegenerative conditions. Indeed, alterations to microglial morphology and function have been recognized as a part of normal aging. Here, we sought to assess the effects of age on the retinal microglial and macrophage response to acute intraocular pressure (IOP) elevation. Further, we performed experiments whereby bone marrow from young or middle-aged mice was used to reconstitute the bone marrow of whole-body irradiated 12 month old mice. Bone marrow chimeric mice then underwent cannulation and IOP elevation 8 weeks after whole-body irradiation and bone marrow transplantation in order to determine whether the age of bone marrow alters the macrophage response to retinal injury. Our data show retinal macrophage reactivity and microglial morphological changes were enhanced in older mice when compared to younger mice in response to injury. When IOP elevation was performed after whole-body irradiation and bone marrow rescue, we noted subretinal macrophage accumulation and glial reactivity was reduced compared to non-irradiated mice that had also undergone IOP elevation. This effect was evident in both groups of chimeric mice that had received either young or middle-aged bone marrow, suggesting irradiation itself may alter the macrophage and glial response to injury rather than the age of bone marrow.

Exosomes Secreted by Young Mesenchymal Stem Cells Promote New Bone Formation During Distraction Osteogenesis in Older Rats.

Distraction osteogenesis (DO) is a clinically effective procedure to regenerate large bone defects. However, the treatment duration is undesirably lengthy, especially in elderly patients. Exosomes derived from mesenchymal stem cells (MSC-Exos) could exert the beneficial effects while avoiding the possible complications of stem cell transplantation. This study aimed to evaluate the effects of MSC-Exos on bone regeneration during DO in older rats. Exosomes were isolated from the supernatants of young bone marrow mesenchymal stem cells (BMSCs) through ultra-centrifugation, and characterized using transmission electron microscopy, western blot, and tunable resistive pulse sensing analysis. The effects of MSC-Exos on the proliferation and differentiation of older BMSCs were evaluated using CCK-8 assay, ALP and ARS staining, and qRT-PCR. Unilateral tibial DO model was established on older Sprague-Dawley rats and MSC-Exos or phosphate buffer saline was locally injected into the distraction gaps after distraction weekly. Bone regeneration were evaluated using X-ray, Micro-CT, mechanical test, and histological staining. The MSC-Exos were round or cup-shaped vesicles ranging from 60 to 130nm in diameter and expressed markers including CD9, CD63, and TSG101. The in vitro results indicated that MSC-Exos could enhance the proliferation and osteogenic differentiation of older BMSCs. Bone regeneration was markedly accelerated in rats treated with MSC-Exos according to the results of X-ray, micro-CT, and histological analysis. The distracted tibias from the MSC-Exos group also demonstrated better mechanical properties. These results suggest that MSC-Exos promote DO-mediated bone regeneration in older rats through enhancing the proliferation and osteogenic capacity of BMSCs.

Rectification of radiotherapy-induced cognitive impairments in aged mice by reconstituted Sca-1+ stem cells from young donors

BackgroundRadiotherapy is widely used and effective for treating brain tumours, but inevitably impairs cognition as it arrests cellular processes important for learning and memory. This is particularly evident in the aged brain with limited regenerative capacity, where radiation produces irreparable neuronal damage and activation of neighbouring microglia. The latter is responsible for increased neuronal death and contributes to cognitive decline after treatment. To date, there are few effective means to prevent cognitive deficits after radiotherapy.MethodsHere we implanted hematopoietic stem cells (HSCs) from young or old (2- or 18-month-old, respectively) donor mice expressing green fluorescent protein (GFP) into old recipients and assessed cognitive abilities 3 months post-reconstitution.ResultsRegardless of donor age, GFP+ cells homed to the brain of old recipients and expressed the macrophage/microglial marker, Iba1. However, only young cells attenuated deficits in novel object recognition and spatial memory and learning in old mice post-irradiation. Mechanistically, old recipients that received young HSCs, but not old, displayed significantly greater dendritic spine density and long-term potentiation (LTP) in CA1 neurons of the hippocampus. Lastly, we found that GFP+/Iba1+ cells from young and old donors were differentially polarized to an anti- and pro-inflammatory phenotype and produced neuroprotective factors and reactive nitrogen species in vivo, respectively.ConclusionOur results suggest aged peripherally derived microglia-like cells may exacerbate cognitive impairments after radiotherapy, whereas young microglia-like cells are polarized to a reparative phenotype in the irradiated brain, particularly in neural circuits associated with rewards, learning, and memory. These findings present a proof-of-principle for effectively reinstating central cognitive function of irradiated brains with peripheral stem cells from young donor bone marrow.

Multiscale bone quality analysis in osteoarthritic knee joints reveal a role of the mechanosensory osteocyte network in osteophytes

Osteophytes - bony outgrowths on joint structures - are found in healthy individuals but are specifically present in late osteoarthritis (OA). Osteophyte development and function is not well understood, yet biomechanical stimuli are thought to be critical. Bone adapts to mechanical forces via the cellular network of osteocytes. The involvement of osteocytes in osteophyte formation and maturation has not been unravelled. Forty-three osteophytes from tibias of 23 OA patients (65 ± 9 years) were analysed. The trabecular bone structure of osteophytes presented with fewer trabeculae of lower bone mineral density compared to subchondral bone. We identified 40% early stage and 60% late stage osteophytes that significantly differed in their trabecular bone characteristics. Osteophyte bone revealed a higher number of osteocytes and a lower number of empty osteocyte lacunae per bone area than the subchondral bone. We found that OA osteophytes consist of younger bone material comprised of woven and lamellar bone with the capacity to develop into a late stage osteophyte potentially via the involvement of the osteocyte network. Our analysis of OA osteophytes implies a transition from woven to lamellar bone as in physiological bone growth within a pathological joint. Therefore, osteophyte development and growth present a valuable research subject when aiming to investigate the osteogenic signalling cascade.

Influence of mesenchymal stem cell-derived extracellular vesicles in vitro and their role in ageing

IntroductionThis study assessed whether mesenchymal stem cell (MSC)-derived extracellular vesicles influenced ageing and pluripotency markers in cell cultures where they are added.MethodsMSC-derived extracellular vesicles from old and young rat bone marrows were isolated by ultracentrifugation and were characterised by western blotting, nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). They were added to young and old MSC cultures. Real-time quantitative reverse transcription polymerase chain reactions and western blot analysis were performed to check the markers of ageing (vinculin and lamin A), pluripotency markers (Nanog and Oct4) and components of the mTOR signalling pathway (Rictor, Raptor, AKT and mTOR) in these cell populations. Subsequently, microRNA (miR)-188-3p expression was transiently inhibited in young MSCs to demonstrate the influence of mTOR2 on MSC ageing.ResultsIncubation with young MSC-derived extracellular vesicles decreased the levels of ageing markers and components of the mTOR pathway and increased the pluripotency markers from old MSC populations. By contrast, incubation of young MSCs with old MSC-derived extracellular vesicles generated the reverse effects. Inhibition of miR-188-3p expression in young MSCs produced extracellular vesicles that when incubated with old MSCs produced an increase in the levels of Rictor, as well as a decrease of phosphor-AKT, as indicated by a significant decrease in beta-galactosidase staining.ConclusionsMSC-derived extracellular vesicles affected the behaviour of MSC cultures, based on their composition, which could be modified in vitro. These experiments represented the basis for the development of new therapies against ageing-associated diseases using MSC-derived extracellular vesicles.

LPS-induced premature osteocyte senescence: Implications in inflammatory alveolar bone loss and periodontal disease pathogenesis.

Cellular senescence is associated with inflammation and extracellular matrix tissue remodeling through the secretion of proteins termed the senescence-associated secretory phenotype (SASP). Although osteocyte senescence in older individuals in the skeleton is well recognized, whether young alveolar osteocytes can also become senescent is unknown. This is potentially important in the context of periodontal disease, which is an inflammatory condition caused by a gradual change from symbiotic to pathogenic oral microflora that can lead to tooth loss. Our aim was to identify whether senescent osteocytes accumulate in young alveolar bone and whether bacterial-derived lipopolysaccharide (LPS) can influence cellular senescence in alveolar bone. An osteocyte-enriched cell population isolated from alveolar bone expressed increased levels of the known senescence marker p16Ink4a, as well as select SASP markers known to be implicated alveolar bone resorption (Icam1, Il6, Il17, Mmp13 and Tnfα), compared to ramus control cells. Increased senescence of alveolar bone osteocytes was also observed in vivo using the senescence-associated distension of satellites (SADS) assay and increased γH2AX, a marker of DNA damage associated with senescent cells. To approximate a bacterial infection in vitro, alveolar osteocytes were treated with LPS. We found increased expression of various senescence and SASP markers, increased γH2AX staining, increased SA-β-Gal activity and the redistribution of F-actin leading to a larger and flattened cell morphology, all hallmarks of cellular senescence. In conclusion, our data suggests a model whereby bacterial-derived LPS stimulates premature alveolar osteocyte senescence, which in combination with the resultant SASP, could potentially contribute to the onset of alveolar bone loss.