Ann-Kristin Picke

Effects of parathyroid hormone on bone mass, bone strength, and bone regeneration in male rats with type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM) is associated with increased skeletal fragility and impaired fracture healing. Intermittent PTH therapy increases bone strength; however, its skeletal and metabolic effects in diabetes are unclear. We assessed whether PTH improves skeletal and metabolic function in rats with T2DM. Subcritical femoral defects were created in diabetic fa/fa and nondiabetic +/+ Zucker Diabetic Fatty (ZDF) rats and internally stabilized. Vehicle or 75 μg/kg/d PTH(1-84) was sc administered over 12 weeks. Skeletal effects were evaluated by μCT, biomechanical testing, histomorphometry, and biochemical markers, and defect regeneration was analyzed by μCT. Glucose homeostasis was assessed using glucose tolerance testing and pancreas histology. In diabetic rats, bone mass was significantly lower in the distal femur and vertebrae, respectively, and increased after PTH treatment by up to 23% in nondiabetic and up to 18% in diabetic rats (P < .0001). Diabetic rats showed 23% lower ultimate strength at the spine (P < .0005), which was increased by PTH by 36% in normal and by 16% in diabetic rats (P < .05). PTH increased the bone formation rate by 3-fold in normal and by 2-fold in diabetic rats and improved defect regeneration in normal and diabetic rats (P < .01). PTH did not affect serum levels of undercarboxylated osteocalcin, glucose tolerance, and islet morphology. PTH partially reversed the adverse skeletal effects of T2DM on bone mass, bone strength, and bone defect repair in rats but did not affect energy metabolism. The positive skeletal effects were generally more pronounced in normal compared with diabetic rats.

Bone defect regeneration and cortical bone parameters of type 2 diabetic rats are improved by insulin therapy.

Zucker Diabetic Fatty (ZDF) rats represent an established model of type 2 diabetes mellitus (T2DM) and display several features of human diabetic bone disease, including impaired osteoblast function, decreased bone strength, and delayed bone healing. Here, we determined whether glycemic control by insulin treatment prevents skeletal complications associated with diabetes. Subcritical femur defects were created in diabetic (fa/fa) and non-diabetic (+/+) ZDF rats. Diabetic rats were treated once daily with long-lasting insulin glargin for 12weeks for glycemic control. Insulin treatment successfully maintained serum levels of glycated hemoglobin, while untreated diabetic rats showed a 2-fold increase. Trabecular and cortical bone mass measured by μCT were decreased in diabetic rats. Insulin treatment increased bone mass of the cortical, but not of the trabecular bone compartment. Dynamic histomorphometry revealed a lower bone formation rate at the trabecular and periosteal cortical bone in diabetic animals and decreased serum procollagen type 1 N-terminal propeptide (P1NP, -49%) levels. Insulin treatment partially improved these parameters. In T2DM, serum levels of tartrate-resistant acid phosphatase (TRAP, +32%) and C-terminal telopeptide (CTX, +49%) were increased. Insulin treatment further elevated TRAP levels, but did not affect CTX levels. While diabetes impaired bone defect healing, glycemic control with insulin fully reversed these negative effects. In conclusion, insulin treatment reversed the adverse effects of T2DM on bone defect regeneration in rats mainly by improving osteoblast function and bone formation. This article is part of a Special Issue entitled Bone and diabetes.

Structural and functional insights into sclerostin-glycosaminoglycan interactions in bone.

In order to improve bone defect regeneration, the development of new adaptive biomaterials and their functional and biological validation is warranted. Glycosaminoglycans (GAGs) are important extracellular matrix (ECM) components in bone and may display osteogenic properties that are potentially useful for biomaterial coatings. Using hyaluronan (HA), chondroitin sulfate (CS) and chemically modified highly sulfated HA and CS derivatives (sHA3 and sCS3; degree of sulfation ∼3), we evaluated how GAG sulfation modulates Wnt signaling, a major regulator of osteoblast, osteoclast and osteocyte biology. GAGs were tested for their capability to bind to sclerostin, an inhibitor of Wnt signaling, using surface plasmon resonance and molecular modeling to characterize their interactions. GAGs bound sclerostin in a concentration- and sulfate-dependent manner at a common binding region. These findings were confirmed in an LRP5/sclerostin interaction study and an inxa0vitro model of Wnt activation. Here, pre-incubation of sclerostin with different GAGs led to a sulfate- and dose-dependent loss of its bioactivity. Using GAG-biotin derivatives in a competitive ELISA approach sclerostin was shown to be the preferred binding partner over Wnt3a. In conclusion, highly sulfated GAGs might control bone homeostasis via interference with sclerostin/LRP5/6 complex formation. Whether these properties can be utilized to improve bone regeneration needs to be validated inxa0vivo.

Effects of parathyroid hormone on cortical porosity, non-enzymatic glycation and bone tissue mechanics in rats with type 2 diabetes mellitus

Type 2 diabetes mellitus increases skeletal fragility; however, the contributing mechanisms and the efficacy of bone-forming agents are unclear. We studied diabetes and parathyroid hormone (PTH) treatment effects on cortical porosity (Ct.Po), non-enzymatic glycation (NEG) and bone mechanics in Zucker diabetic fatty (ZDF) rats. Eleven-week old ZDF diabetic (DB) and non-diabetic (ND) rats were given 75μg/kg PTH (1-84) or vehicle 5days per week over 12weeks. The right femora and L4 vertebrae were excised, micro-CT scanned, and tested in 3-point bending and uniaxial compression, respectively. NEG of the samples was determined using fluorescence. Diabetes increased Ct.Po (vertebra (vert): +40.6%, femur (fem): +15.5% vs. ND group, p<0.05) but had no effect on NEG. PTH therapy reduced vertebral NEG in the ND animals only (-73% vs untreated group, p<0.05), and increased femoral NEG in the DB vs. ND groups (+63%, p<0.05). PTH therapy had no effect on Ct.Po. Diabetes negatively affected bone tissue mechanics where reductions in vertebral maximum strain (-22%) and toughness (-42%) were observed in the DB vs. ND group (p<0.05). PTH improved maximum strain in the vertebra of the ND animals (+21%, p<0.05) but did not have an effect in the DB group. PTH increased femoral maximum strain (+21%) and toughness (+28%) in ND and decreased femoral maximum stress (-13%) and toughness (-27%) in the DB animals (treated vs. untreated, p<0.05). Ct.Po correlated negatively with maximum stress (fem: R=-0.35, p<0.05, vert: R=-0.57, p<0.01), maximum strain (fem: R=-0.35, p<0.05, vert: R=-0.43, p<0.05) and toughness (fem: R=-0.34, p<0.05, vert: R=-0.55, p<0.01), and NEG correlated negatively with toughness at the femur (R=-0.34, p<0.05) and maximum strain at the vertebra (R=-0.49, p<0.05). Diabetes increased cortical porosity and reduced bone mechanics, which were not improved with PTH treatment. PTH therapy alone may worsen diabetic bone mechanics through formation of new bone with high AGEs cross-linking. Optimal treatment regimens must address both improvements of bone mass and glycemic control in order to successfully reduce diabetic bone fragility. This article is part of a Special Issue entitled Bone and diabetes.

Sulfated hyaluronan improves bone regeneration of diabetic rats by binding sclerostin and enhancing osteoblast function.

Bone fractures in patients with diabetes mellitus heal poorly and require innovative therapies to support bone regeneration. Here, we assessed whether sulfated hyaluronan included in collagen-based scaffold coatings can improve fracture healing in diabetic rats. Macroporous thermopolymerized lactide-based scaffolds were coated with collagen including non-sulfated or sulfated hyaluronan (HA/sHA3) and inserted into 3xa0mm femoral defects of non-diabetic and diabetic ZDF rats. After 12 weeks, scaffolds coated with collagen/HA or collagen/sHA3 accelerated bone defect regeneration in diabetic, but not in non-diabetic rats as compared to their non-coated controls. At the tissue level, collagen/sHA3 promoted bone mineralization and decreased the amount of non-mineralized bone matrix. Moreover, collagen/sHA3-coated scaffolds from diabetic rats bound more sclerostin inxa0vivo than the respective controls. Binding assays confirmed a high binding affinity of sHA3 to sclerostin. Inxa0vitro, sHA3 induced BMP-2 and lowered the RANKL/OPG expression ratio, regardless of the glucose concentration in osteoblastic cells. Both sHA3 and high glucose concentrations decreased the differentiation of osteoclastic cells. In summary, scaffolds coated with collagen/sHA3 represent a potentially suitable biomaterial to improve bone defect regeneration in diabetic conditions. The underlying mechanism involves improved osteoblast function and binding sclerostin, a potent inhibitor of Wnt signaling and osteoblast function.

Thy-1 (CD90) promotes bone formation and protects against obesity

Thy-1 on mesenchymal stem cells promotes osteogenesis while inhibiting adipogenesis and obesity. Stem cells’ balancing act Mesenchymal stem cells (MSCs) differentiate into multiple cell types. Picke et al. investigated how MSC differentiation is regulated to maintain homeostasis between the bone and fat lineages. Genetic deletion of Thy-1, a protein expressed on multiple cell types including MSCs, prevented MSC differentiation into osteoblasts but promoted differentiation into adipocytes. Thy-1–deficient mice had increased body fat and decreased bone mass. High-fat diet induced obesity in wild-type mice and concurrent reduction in bone formation, which was associated with decreased Thy-1 expression on MSCs. Obese human subjects and subjects with osteoporosis showed reductions in serum soluble Thy-1. This study suggests that Thy-1 regulates the balance between bone and fat lineages, with possible implications for bone and metabolic disorders. Osteoporosis and obesity result from disturbed osteogenic and adipogenic differentiation and present emerging challenges for our aging society. Because of the regulatory role of Thy-1 in mesenchyme-derived fibroblasts, we investigated the impact of Thy-1 expression on mesenchymal stem cell (MSC) fate between osteogenic and adipogenic differentiation and consequences for bone formation and adipose tissue development in vivo. MSCs from Thy-1–deficient mice have decreased osteoblast differentiation and increased adipogenic differentiation compared to MSCs from wild-type mice. Consistently, Thy-1–deficient mice exhibited decreased bone volume and bone formation rate with elevated cortical porosity, resulting in lower bone strength. In parallel, body weight, subcutaneous/epigonadal fat mass, and bone fat volume were increased. Thy-1 deficiency was accompanied by reduced expression of specific Wnt ligands with simultaneous increase of the Wnt inhibitors sclerostin and dickkopf-1 and an altered responsiveness to Wnt. We demonstrated that disturbed bone remodeling in osteoporosis and dysregulated adipose tissue accumulation in patients with obesity were mirrored by reduced serum Thy-1 concentrations. Our findings provide new insights into the mutual regulation of bone formation and obesity and open new perspectives to monitor and to interfere with the dysregulated balance of adipogenesis and osteogenesis in obesity and osteoporosis.

Differential effects of high-fat diet and exercise training on bone and energy metabolism

Bone microarchitecture and strength are impaired by obesity and physical inactivity, but the underlying molecular regulation of bone metabolism in response to these factors is not well understood. Therefore, we analyzed bone and energy metabolism in male mice fed a high-fat or standard chow diet for 12u202fweeks with or without free access to running wheels. High-fat diet (HFD) mimicked the human condition of obesity and insulin resistance, including symptoms such as elevated serum glucose and insulin levels and reduced insulin-stimulated glucose uptake into muscle and adipose tissue. Interestingly, HFD also decreased (-44%) glucose uptake into bone marrow. Bone mass was reduced (-45%) by HFD due to a diminished (-45%) bone remodeling rate. Bone matrix quality aspects, such as biomechanical stability, were additionally decreased. Concurrently, the bone marrow adiposity increased (+63%) in response to a HFD. Further, we detected elevated expression of the Wnt signaling inhibitor dickkopf-1 (Dkk-1, +42%) in mice fed a HFD, but this was not reflected in serum samples obtained from obese humans. In mice, exercise attenuated the adverse effects of HFD by reversing the glucose uptake into bone marrow, improving the bone mass and bone matrix quality while decreasing the bone marrow adiposity. This data shows that exercise prevents some, but not all of the negative effects of HFD on bone health and suggests that insulin signaling in bone marrow and Dkk-1 signaling may be involved in the pathogenesis of bone loss induced by HFD.

Thy-1 Deficiency Augments Bone Loss in Obesity by Affecting Bone Formation and Resorption

Healthy bone remodeling results from a balanced bone formation and bone resorption realized by bone-forming osteoblasts and bone-resorbing osteoclasts, respectively. Recently, Thy-1 (CD90) was identified as positive regulator of osteoblast differentiation and activation, thus, promoting bone formation while concurrently inhibiting adipogenesis and obesity in mice. Additionally, Thy-1 did not affect bone resorption. An obesity-related co-morbidity that is increasing in prevalence is a disturbed bone formation resulting in an increased fracture risk. The underlying mechanisms of obesity-induced bone alterations are not yet fully elucidated and therefore therapy options for efficient bone-anabolic treatments are limited. Therefore, we investigated the impact of Thy-1 on bone metabolism under obese conditions. Indeed, high fat diet (HFD) induced obese mice lacking Thy-1 (Thy-1−/−) showed increased body fat mass compared to wildtype (WT) mice while bone mass (−38%) and formation (−57%) were decreased as shown by micro-computed tomography (μCT) measurement, histological analysis, and fourier-transform infrared spectroscopy (FTIR). Interestingly, under obese conditions, lack of Thy-1 affected both osteoblast and osteoclast function. Number (−30%) and activity of osteoblasts were decreased in obese Thy-1−/− mice while osteoclast number (+39%) and activity were increased. Facilitated bone marrow fat accumulation (+56%) in obese Thy-1−/− mice compared to obese WT mice was associated with upregulated tumor necrosis factor α (Tnfα, +46%) and colony stimulating factor 1 receptor (Csf1r) expression, strong promoters of osteoclast differentiation. Moreover, lack of Thy-1 was accompanied by a reduction of osteoprotegerin (Tnfrsf11b) expression (−36%), an inhibitor of osteoclast differentiation. Altered Tnfα, Csf1r, and Tnfrsf11b expression might be responsible for elevated osteoclast activity in obese Thy-1-deficient mice. In summary, our findings show that lack of Thy-1 promotes obesity under HFD conditions while concurrently decreasing bone mass and formation. Mechanistic studies revealed that under obese conditions lack of Thy-1 impairs both bone formation and bone resorption.

Effects of insulin therapy on porosity, non-enzymatic glycation and mechanical competence in the bone of rats with type 2 diabetes mellitus.

Type 2 diabetes mellitus increases skeletal fragility; however, the contributing mechanisms and optimal treatment strategies remain unclear. We studied the effects of diabetes and insulin therapy on non-enzymatic glycation (NEG), cortical porosity (Ct.Po) and biomechanics of the bone tissue in Zucker Diabetic Fatty (ZDF) rats. Eleven-week old ZDF diabetic and non-diabetic rats were given insulin to achieve glycaemic control or vehicle seven days per week over twelve weeks (insulin dose adapted individually 0.5 international units (IU) at week 1 to 13.0IU at week 12). The right femora were excised, micro-CT scanned, and tested in 3-point bending to measure biomechanics. NEG of the midshaft was determined from bulk fluorescence. Diabetes led to increased NEG (+50.1%, p=0.001) and Ct.Po (+22.9%, p=0.004), as well as to reduced mechanical competence (max. stress: -14.2%, p=0.041, toughness: -29.7%, p=0.016) in the bone tissue. NEG and Ct.Po both correlated positively to serum glucose (NEG: R(2)=0.41, p<0.001, Ct.Po: R(2)=0.34, p=0.003) and HbA1c (NEG: R(2)=0.42, p<0.001, Ct.Po: R(2)=0.28, p=0.008) levels, while NEG correlated negatively with bone biomechanics (elastic modulus: R(2)=0.21, p=0.023, yield stress: R(2)=0.17, p=0.047). Twelve weeks of insulin therapy had no significant effect on NEG or Ct.Po, and was unable to improve the mechanical competence of the bone tissue. A reduction of mechanical competence was observed in the bone tissue of the diabetic rats, which was explained in part by increased collagen NEG. Twelve weeks of insulin therapy did not alter NEG, Ct.Po or bone biomechanics. However, significant correlations between NEG and serum glucose and HbA1c were observed, both of which were reduced with insulin therapy. This suggests that a longer duration of insulin therapy may be required to reduce the NEG of the bone collagen and restore the mechanical competence of diabetic bone.