Hamann C, Goettsch C, Mettelsiefen J, Henkenjohann V, Rauner M, Hempel U, Bernhardt R, Fratzl-Zelman N, Roschger P, Rammelt S, Günther KP, Hofbauer LC. Delayed bone regeneration and low bone mass in a rat model of insulin-resistant type 2 diabetes mellitus is due to impaired osteoblast function. Am J Physiol Endocrinol Metab 301: E1220 -E1228, 2011. First published September 6, 2011; doi:10.1152/ajpendo.00378.2011.-Patients with diabetes mellitus have an impaired bone metabolism; however, the underlying mechanisms are poorly understood. Here, we analyzed the impact of type 2 diabetes mellitus on bone physiology and regeneration using Zucker diabetic fatty (ZDF) rats, an established rat model of insulin-resistant type 2 diabetes mellitus. ZDF rats develop diabetes with vascular complications when fed a Western diet. In 21-wk-old diabetic rats, bone mineral density (BMD) was 22.5% (total) and 54.6% (trabecular) lower at the distal femur and 17.2% (total) and 20.4% (trabecular) lower at the lumbar spine, respectively, compared with nondiabetic animals. BMD distribution measured by backscattered electron imaging postmortem was not different between diabetic and nondiabetic rats, but evaluation of histomorphometric indexes revealed lower mineralized bone volume/tissue volume, trabecular thickness, and trabecular number. Osteoblast differentiation of diabetic rats was impaired based on lower alkaline phosphatase activity (Ϫ20%) and mineralized matrix formation (Ϫ55%). In addition, the expression of the osteoblast-specific genes bone morphogenetic protein-2, RUNX2, osteocalcin, and osteopontin was reduced by 40 -80%. Osteoclast biology was not affected based on tartrate-resistant acidic phosphatase staining, pit formation assay, and gene profiling. To validate the implications of these molecular and cellular findings in a clinically relevant model, a subcritical bone defect of 3 mm was created at the left femur after stabilization with a four-hole plate, and bone regeneration was monitored by X-ray and microcomputed tomography analyses over 12 wk. While nondiabetic rats filled the defects by 57%, diabetic rats showed delayed bone regeneration with only 21% defect filling. In conclusion, we identified suppressed osteoblastogenesis as a cause and mechanism for low bone mass and impaired bone regeneration in a rat model of type 2 diabetes mellitus. bone defect; bone matrix mineralization; bone regeneration; type 2 diabetes mellitus; osteoblast; osteoclast DIABETES MELLITUS TYPE 2 AND the associated metabolic syndrome have become epidemic clinical and economic health problems (1). Morbidity and mortality of diabetes mellitus are determined by vascular complications, including cardiovascular disease, retinopathy, nephropathy, and polyneuropathy (11). Skeletal sequelae of long-standing diabetes mellitus include Charcot neuroarthropathy and the diabetic foot syndrome, which may require amputation. More recently, osteoporosis with an increased risk of fragility fractures has emerged as a complication in patients with long-stan...

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Type 2 diabetes mellitus results in increased risk of fracture and delayed fracture healing. ZDF fa/fa rats are an established model of type 2 diabetes mellitus with low bone mass and delayed bone healing. We tested whether a sclerostin-neutralizing antibody (Scl-AbVI) would reverse the skeletal deficits of diabetic ZDF rats. Femoral defects of 3 mm were created in 11-week-old diabetic ZDF fa/fa and nondiabetic ZDF þ/þ rats and stabilized by an internal plate. Saline or 25 mg/kg Scl-AbVI was administered subcutaneously (s.c.) twice weekly for 12 weeks (n ¼ 9-10/group). Bone mass and strength were assessed using pQCT, micro-computed tomography (mCT), and biomechanical testing. Bone histomorphometry was used to assess bone formation, and the filling of the bone defect was analyzed by mCT. Diabetic rats displayed lower spinal and femoral bone mass compared to nondiabetic rats, and Scl-AbVI treatment significantly enhanced bone mass of the femur and the spine of diabetic rats (p < 0.0001). Scl-AbVI also reversed the deficit in bone strength in the diabetic rats, with 65% and 89% increases in maximum load at the femoral shaft and neck, respectively (p < 0.0001). The lower bone mass in diabetic rats was associated with a 65% decrease in vertebral bone formation rate, which Scl-AbVI increased by sixfold, consistent with a pronounced anabolic effect. Nondiabetic rats filled 57% of the femoral defect, whereas diabetic rats filled only 21% (p < 0.05). Scl-AbVI treatment increased defect regeneration by 47% and 74%, respectively (p < 0.05). Sclerostin antibody treatment reverses the adverse effects of type 2 diabetes mellitus on bone mass and strength, and improves bone defect regeneration in rats. ß

Purpose: Type-I collagen, the major structural protein in bone, has beneficial properties regarding bone regeneration. Little is known about the potential effects of collagen coating on orthopedic implants. method^ 3 to 6 @cm2 of lyophilized type-I collagen was absorbed on titanium rods. Six coated and uncoated pins of 0.9 mm diameter were inserted into the tibia of adult male Wistar rats for 1, 2, 4, 7, 14, and 28 days. Specimens were embedded in methacrylate-based Technovit 9100N resin. From one portion cutting and grinding sections were obtained. The implant was removed from the other half that was depolymerized, sectioned, and mounted for immunohistochemistry.Results: At day 4, the interface around the collagen-coated implants displayed a granulation tissue with higher numbers of cathepsin D-positive mononucleated cells compared to the uncoated implants @ < 0.05). Active osteoblasts, reactive for osteopontin, were increased around the collagen-coated pins at day 4 and 7 (p < 0.01). After 28 days of implantation, direct bone contact averaged 74.9% around the collagen-coated implants and 62.1% around uncoated implants (NS). The amount of newly formed bone averaged 76.3% around the collagen-coated pins and 67.8% around the uncoated pins (NS). The histomorphometric findings were confirmed by SRpCT in two specimens. Conclusions:The earlier observation of mononuclear phagocytozing cells and the earlier and higher expression of bone-specific matrix proteins suggest an increased early bone remodeling around titanium pins through collagen coating. A tendency towards increased bone formation was observed around the coated implants.

Histological imaging is still considered the gold standard for analysing bone formation around metallic implants. Generally, a limited number of histological sections per sample are used for the approximation of mean values of peri-implant bone formation. In this study we compared statistically the results of bone-implant contact (BIC) and bone-implant volume (BIV) obtained by histological sections, with those obtained by X-ray absorption images from synchrotron radiation micro-computed tomography (SRμCT) using osseointegrated screw-shaped implants from a mini-pig study. Comparing the BIC results of 3-4 histological sections per implant sample with the appropriate 3-4 SRμCT slices showed a non-signifi cant difference of 1.9 % (p = 0.703). The contact area assessed by the whole 3D information from the SRμCT measurement in comparison to the histomorphometric results showed a non-signifi cant difference in BIC of 4.9 % (p = 0.171). The amount of the bone-implant volume in the histological sections and the appropriate SRμCT slices showed a nonsignifi cant difference by only 1.4 % (p = 0.736) and also remains non-signifi cant with 2.6 % (p = 0.323) using the volumetric SRμCT information. We conclude that for a clinical evaluation of implant osseointegration with histological imaging at least 3-4 sections per sample are suffi cient to represent the BIC or BIV for a sample. Due to the fact that in this study we have found a signifi cant intra-sample variation in BIC of up to ± 35 % the selection of only one or two histological sections per sample may strongly infl uence the determined BIC.