Type 2 Diabetes Mellitus Is Associated With Better Bone Microarchitecture But Lower Bone Material Strength and Poorer Physical Function in Elderly Women: A Population‐Based Study

Type 2 diabetes mellitus (T2DM) is associated with an increased risk of fractures according to several studies. The underlying mechanisms remain unclear, although small case‐control studies indicate poor quality of the cortical bone. We have studied a population‐based sample of women aged 75 to 80 years in Gothenburg, randomly invited from the population register. Areal bone mineral density (aBMD) was measured by dual‐energy X‐ray absorptiometry (Hologic Discovery A), bone microarchitecture by high‐resolution peripheral quantitative computed tomography (HR‐pQCT; ExtremeCT from Scanco Medical AG), and reference point indentation was performed with Osteoprobe (Active Life Scientific). Women with T2DM (n = 99) had higher aBMD compared to controls (n = 954). Ultradistal tibial and radial trabecular bone volume fraction (+11% and +15%, respectively), distal cortical volumetric BMD (+1.6% and +1.7%), cortical area (+11.5% and +9.3%), and failure load (+7.7% and +12.9%) were higher in diabetics than in controls. Cortical porosity was lower (mean ± SD: 1.5% ± 1.1% versus 2.0% ± 1.7%, p = 0.001) in T2DM in the distal radius but not in the ultradistal radius or the tibia. Adjustment for covariates (age, body mass index, glucocorticoid treatment, smoking, physical activity, calcium intake, bone‐active drugs) eliminated the differences in aBMD but not in HR‐pQCT bone variables. However, bone material strength index (BMSi) by reference point indentation was lower in T2DM (74.6 ± 7.6 versus 78.2 ± 7.5, p < 0.01), also after adjustment, and women with T2DM performed clearly worse in measures of physical function (one leg standing: –26%, 30‐s chair‐stand test: –7%, timed up and go: +12%, walking speed: +8%; p < 0.05‐0.001) compared to controls. In conclusion, we observed a more favorable bone microarchitecture but no difference in adjusted aBMD in elderly women with T2DM in the population compared to nondiabetics. Reduced BMSi and impaired physical function may explain the increased fracture risk in T2DM. © 2016 American Society for Bone and Mineral Research.     

Muscle and Bone Impairment in Children With Marfan Syndrome: Correlation With Age and FBN1 Genotype

Marfan syndrome (MFS) is a rare connective tissue disorder caused by mutation in the gene encoding the extracellular matrix protein fibrillin‐1 (FBN1), leading to transforming growth factor‐beta (TGF‐β) signaling dysregulation. Although decreased axial and peripheral bone mineral density (BMD) has been reported in adults with MFS, data about the evolution of bone mass during childhood and adolescence are limited. The aim of the present study was to evaluate bone and muscle characteristics in children, adolescents, and young adults with MFS. The study population included 48 children and young adults (22 girls) with MFS with a median age of 11.9 years (range 5.3 to 25.2 years). The axial skeleton was analyzed at the lumbar spine using dual‐energy X‐ray absorptiometry (DXA), whereas the appendicular skeleton (hand) was evaluated using the BoneXpert system (with the calculation of the Bone Health Index). Muscle mass was measured by DXA. Compared with healthy age‐matched controls, bone mass at the axial and appendicular levels and muscle mass were decreased in children with MFS and worsened from childhood to adulthood. Vitamin D deficiency (<50 nmol/L) was found in about a quarter of patients. Serum vitamin D levels were negatively correlated with age and positively correlated with lumbar spine areal and volumetric BMD. Lean body mass (LBM) Z‐scores were positively associated with total body bone mineral content (TB‐BMC) Z‐scores, and LBM was an independent predictor of TB‐BMC values, suggesting that muscle hypoplasia could explain at least in part the bone loss in MFS. Patients with a FBN1 premature termination codon mutation had a more severe musculoskeletal phenotype than patients with an inframe mutation, suggesting the involvement of TGF‐β signaling dysregulation in the pathophysiologic mechanisms. In light of these results, we recommend that measurement of bone mineral status should be part of the longitudinal clinical investigation of MFS children. © 2015 American Society for Bone and Mineral Research.     

Adverse Effects of Osteocytic Constitutive Activation of ß‐Catenin on Bone Strength and Bone Growth

The activation of the canonical Wnt/β‐catenin signaling pathway in both mesenchymal stem cells and osteoblasts has been demonstrated to increase bone mass, showing promise for the treatment of low bone volume conditions such as osteoporosis. However, the possible side effects of manipulating this pathway have not been fully addressed. Previously, we reported that the constitutive activation of ß‐catenin in osteoblasts impaired vertebral linear growth. In the present study, β‐catenin was constitutively activated in osteocytes by crossing Catnb+/lox(exon 3) mice with dentin matrix protein 1(DMP1)‐Cre transgenic mice, and the effects of this activation on bone mass, bone growth and bone strength were then observed. DMP1‐Cre was found to be predominantly expressed in osteocytes, with weak expression in a small portion of osteoblasts and growth plate chondrocytes. After the activation, the cancellous bone mass was dramatically increased, almost filling the entire bone marrow cavity in long bones. However, bone strength decreased significantly. Thinner and more porous cortical bone along with impaired mineralization were responsible for the decrease in bone strength. Furthermore, the mice showed shorter stature with impaired linear growth of the long bones. Moreover, the concentration of serum phosphate decreased significantly after the activation of ß‐catenin, and a high inorganic phosphate (Pi) diet could partially rescue the phenotype of decreased mineralization level and impaired linear growth. Taken together, the constitutive activation of β‐catenin in osteocytes may increase cancellous bone mass; however, the activation also had adverse effects on bone strength and bone growth. These adverse effects should be addressed before the adoption of any therapeutic clinical application involving adjustment of the Wnt/β‐catenin signaling pathway. © 2015 American Society for Bone and Mineral Research.     

Fluocinolone Acetonide Is a Potent Synergistic Factor of TGF‐β3–Associated Chondrogenesis of Bone Marrow–Derived Mesenchymal Stem Cells for Articular Surface Regeneration

Articular cartilage repair remains a challenging problem. Based on a high‐throughput screening and functional analysis, we found that fluocinolone acetonide (FA) in combination with transforming growth factor beta 3 (TGF‐β3) strongly potentiated chondrogenic differentiation of human bone marrow–derived mesenchymal stem cells (hBMSCs). In an in vivo cartilage defect model in knee joints of immunocompromised mice, transplantation of FA/TGF‐β3–treated hBMSCs could completely repair the articular surface. Analysis of the intracellular pathways revealed that FA enhanced TGF‐β3–induced phosphorylation of Smad2 and Smad3. Additionally, we performed a pathway array and found that FA activates the mTORC1/AKT pathway. Chemical inhibition of mTORC1 with rapamycin substantially suppressed FA effect, and inhibition of AKT completely repressed chondrogenesis of hBMSCs. Inhibition of glucocorticoid receptor with mifepristone also suppressed FA effect, suggesting that FA involves binding to the glucocorticoid receptor. Comparative analysis with other glucocorticoids (triamcinolone acetonide [TA] and dexamethasone [DEX]) revealed the unique ability of FA to repair articular cartilage surgical defects. Analysis of intracellular pathways showed that the mTORC1/AKT pathway and the glucocorticoid receptor was highly activated with FA and TA, but to a lesser extent with DEX. Collectively, these results show a unique ability of FA to enhance TGF‐β3–associated chondrogenesis, and suggest that the FA/TGF‐β3 combination may be used as major inducer of chondrogenesis in vitro. Additionally, FA/TGF‐β3 could be potentially applied in a clinical setting to increase the efficiency of regenerative approaches based on chondrogenic differentiation of stem cells. © 2015 American Society for Bone and Mineral Research.     

Aberrant Activation of TGF‐β in Subchondral Bone at the Onset of Rheumatoid Arthritis Joint Destruction

Rheumatoid arthritis (RA) is an autoimmune disease that often leads to joint destruction. A myriad of drugs targeting the immune abnormalities and downstream inflammatory cascades have been developed, but the joint destruction is not effectively halted. Here we report that aberrant activation of TGF‐β in the subchondral bone marrow by immune response increases osteoprogenitors and uncoupled bone resorption and formation in RA mouse/rat models. Importantly, either systemic or local blockade of TGF‐β activity in the subchondral bone attenuated articular cartilage degeneration in RA. Moreover, conditional deletion of TGF‐β receptor II (Tgfbr2) in nestin‐positive cells also effectively halted progression of RA joint destruction. Our data demonstrate that aberrant activation of TGF‐β in the subchondral bone is involved at the onset of RA joint cartilage degeneration. Thus, modulation of subchondral bone TGF‐β activity could be a potential therapy for RA joint destruction. © 2015 American Society for Bone and Mineral Research     

Transplantation of Haploidentical TcRaß‐Depleted Hematopoietic Cells Allows for Optimal Timing and Sustained Correction of the Metabolic Defect in Children With Infantile Osteopetrosis

In osteopetrosis, osteoclast dysfunction can lead to deafness, blindness, bone marrow failure, and death. Hematopoietic cell transplantation (HCT) is currently the only curative treatment, but outcome remains disappointing. Although a rapid progression toward HCT is detrimental to prevent further progress of disease manifestations, 70% of cases lack an HLA‐matched sibling and require alternative stem cell sources. We present two cases of osteopetrosis that successfully received an HCT with haploidentical TcRαβ‐depleted cells from one of the parents. These cases showed no further disease progression, had restoration of functional osteoclasts, and illustrate this approach to enable prompt HCT with ready available parental donors and rapid and sustained hematological, including osteoclast, recovery. © 2016 American Society for Bone and Mineral Research.     

Activation of Wnt Signaling by Mechanical Loading Is Impaired in the Bone of Old Mice

Aging diminishes bone formation engendered by mechanical loads, but the mechanism for this impairment remains unclear. Because Wnt signaling is required for optimal loading‐induced bone formation, we hypothesized that aging impairs the load‐induced activation of Wnt signaling. We analyzed dynamic histomorphometry of 5‐month‐old, 12‐month‐old, and 22‐month‐old C57Bl/6JN mice subjected to multiple days of tibial compression and corroborated an age‐related decline in the periosteal loading response on day 5. Similarly, 1 day of loading increased periosteal and endocortical bone formation in young‐adult (5‐month‐old) mice, but old (22‐month‐old) mice were unresponsive. These findings corroborated mRNA expression of genes related to bone formation and the Wnt pathway in tibias after loading. Multiple bouts (3 to 5 days) of loading upregulated bone formation–related genes, e.g., Osx and Col1a1, but older mice were significantly less responsive. Expression of Wnt negative regulators, Sost and Dkk1, was suppressed with a single day of loading in all mice, but suppression was sustained only in young‐adult mice. Moreover, multiple days of loading repeatedly suppressed Sost and Dkk1 in young‐adult, but not in old tibias. The age‐dependent response to loading was further assessed by osteocyte staining for Sclerostin and LacZ in tibia of TOPGAL mice. After 1 day of loading, fewer osteocytes were Sclerostin‐positive and, corroboratively, more osteocytes were LacZ‐positive (Wnt active) in both 5‐month‐old and 12‐month‐old mice. However, although these changes were sustained after multiple days of loading in 5‐month‐old mice, they were not sustained in 12‐month‐old mice. Last, Wnt1 and Wnt7b were the most load‐responsive of the 19 Wnt ligands. However, 4 hours after a single bout of loading, although their expression was upregulated threefold to 10‐fold in young‐adult mice, it was not altered in old mice. In conclusion, the reduced bone formation response of aged mice to loading may be due to failure to sustain Wnt activity with repeated loading. © 2016 American Society for Bone and Mineral Research.     

Tracking of Environmental Determinants of Bone Structure and Strength Development in Healthy Boys: An Eight‐Year Follow Up Study on the Positive Interaction Between Physical Activity and Protein Intake From Prepuberty to Mid‐Late Adolescence

High protein (> median:Hprot) vs. moderate (< median:MProt) intake was shown to enhance the positive impact of high physical activity (HPA) on proximal femur BMC/aBMD/Area in healthy prepubertal boys. We tested the hypothesis that this synergistic effect would track and influence bone structure and strength until mid‐adolescence. BMC/aBMD/Area was measured at femoral neck (FN) and total hip (TotHip) by DXA in 176 boys at 7.4 ± 0.4 and 15.2 ± 0.5 years (± SD). Distal tibia (DistTib) microstructure and strength were also assessed at 15.2 years by high‐resolution peripheral computerized tomography (HR‐pQCT) and micro‐finite element analysis (µFEA). The positive impact of HProt vs. MProt on FN and TotHip BMC/aBMD/Area, recorded at 7.4 years remained unabated at 15.2 years. At this age, at DistTib, HProt‐HPA vs. MProt‐HPA was associated (p < 0.001) with larger cross‐sectional area (CSA, mm²), trabecular number (Tb.N, mm^?1) and lower trabecular separation (Tb.Sp, µm). The interaction between physical activity and protein intake was significant for CSA (p = 0.012) and Tb.N (p = 0.043). Under MProt (38.0 ± 6.9 g.d^?1), a difference in PA from 168 ± 40 to 303 ± 54 kcal.d^?1 was associated with greater stiffness (kN/mm) and failure load (N) of +0.16 and +0.14 Z‐score, respectively. In contrast, under HProt (56.2 ± 9.5 g.d^?1), a difference in PA of similar magnitude, from 167 ± 33 to 324 ± 80 kcal.d^?1, was associated with a larger difference in stiffness and failure load of +0.50 and +0.57 Z‐score, respectively. In conclusion, the positive influence of relatively HProt on the impact of HPA on proximal femur macrostructure tracks from prepuberty to mid‐late puberty. At this stage, the impact of HProt on HPA is also associated with microstructural changes that should confer greater mechanical resistance to weight‐bearing bones. These results underscore the importance of protein intake and exercise synergistic interaction in the early prevention of adult osteoporosis. © 2014 American Society for Bone and Mineral Research.     

Postoperative Tendon Loading With Treadmill Running Delays Tendon‐to‐Bone Healing: Immunohistochemical Evaluation in a Murine Rotator Cuff Repair Model

Mechanical stress has an important effect on tendon‐to‐bone healing. The purpose of the present study was to compare tendon‐to‐bone healing in animals exposed to either tendon unloading (botulinum toxin injection) or excessive loading (treadmill running) in a murine rotator cuff repair model. Forty‐eight C57BL/6 mice underwent unilateral supraspinatus tendon detachment and repair. Mice in the unloaded group were injected with botulinum toxin to the supraspinatus muscle. The contralateral shoulder of the unloaded group was used as a control. Mice were euthanized at 1, 2, and 4 weeks after surgery and evaluated with hematoxylin–eosin and immunohistochemical (IHC) staining for Ihh, Gli1, Wnt3a, and β‐catenin. The positive staining area on IHC and the Modified Tendon Maturing Score were measured. The score of the unloaded group was significantly higher (better healing) than that of the treadmill group at 4 weeks. Ihh and the glioma‐associated oncogene homolog 1 (Gli1) positive area in the unloaded group were significantly higher than those of the control group at 1 week. The peak time‐points of the Ihh and Gli1 positive area was 1 week for the unloaded group and 2 weeks for the treadmill group. The Wnt3a positive area in the unloaded group was significantly higher than that of the control group at 2 weeks. The β‐catenin positive area in the unloaded group was significantly higher than that of the treadmill group and the control group at 1 week. Our data indicated that the unloaded group has superior tendon maturation compared to the treadmill running group. Excessive tendon loading may delay the tendon healing process by affecting the activity of Ihh and Wnt/β‐Catenin pathways. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1628–1637, 2019.