Distal Femoral Metaphysis Research Articles

The Anterolateral Complex of the Knee.

Background:Significant controversy exists regarding the anterolateral structures of the knee.Purpose:To determine the layer-by-layer anatomic structure of the anterolateral complex of the knee.Study Design:Descriptive laboratory study.Methods:Twenty fresh-frozen cadaveric knees (age range, 38-56 years) underwent a layer-by-layer dissection to systematically expose and identify the various structures of the anterolateral complex. Quantitative measurements were performed, and each layer was documented with high-resolution digital imaging.Results:The anterolateral complex of the knee consisted of different distinct layers, with the superficial and deep iliotibial band (ITB) representing layer 1. The superficial ITB had a distinct connection to the distal femoral metaphysis and femoral condyle (Kaplan fibers), and the deep layers of the ITB were identified originating at the level of the Kaplan fibers proximally. This functional unit, consisting of the superficial and deep ITB, was reinforced by the capsulo-osseous layer of the ITB, which was continuous with the fascia of the lateral gastrocnemius and biceps femoris muscles. These 3 components of the ITB became confluent distally, and the insertion spanned from the Gerdy tubercle anteriorly to the lateral tibia posteriorly on a small tubercle (lateral tibial tuberosity). Layer 3 consisted of the anterolateral capsule, in which 35% (7/20) of specimens had a discreet mid-third capsular ligament.Conclusion:The anterolateral complex consists of the superficial and deep ITB, the capsulo-osseous layer of the ITB, and the anterolateral capsule. The anterolateral complex is defined by the part of the ITB between the Kaplan fibers proximally and its tibial insertion, which forms a functional unit. A discrete anterolateral ligament was not observed; however, the anterolateral ligament described in recent studies likely refers to the capsulo-osseous layer or the mid-third capsular ligament.Clinical Relevance:The anterolateral knee structures form a complex functional unit. Surgeons should use caution when attempting to restore this intricate structure with extra-articular procedures designed to re-create a single discreet ligament.

The effects of downhill and uphill exercise training on osteogenesis-related factors in ovariectomy-induced bone loss.

[Purpose]Recent evidence suggests that regular exercise training plays a decisive role in maintaining homeostasis and promoting muscle and skeletal formation. However, the effect of downhill exercise training on osteogenesis-related factors is not well understood.[Methods]Thus, we investigated the effect of uphill and downhill training on ovariectomy (OVX)-induced bone loss. After ovary removal, the exercise method performed included uphill (16 m/min, +15°) and downhill training (16 m/min, –15°) for 60 min/day and 5 days/week, respectively, for 8 weeks.[Results]Our results showed that both uphill and downhill training significantly decreased the body weight, total cholesterol, and creatine kinase (CK) levels in the context of OVX-induced bone loss. On the contrary, levels of an osteogenesis indicator, osteocalcin and alkaline phosphatase were elevated. Consequently, the uphill and downhill training reduced OVX- induced bone loss in the distal femoral metaphysis. Likewise, the bone microstructure in OVX-induced bone loss was enhanced upon training. In particular, the inhibition of RANKL-induced osteoclast formation and osteoclast-specific gene expression improved upon downhill training compared to uphill training.[Conclusion]These results suggest that the uphill and downhill exercise types appeared to positively affect the expression of osteogenesis-related factors along with bone density and microstructure. Particularly, the downhill training has more beneficial effects on the maintenance of homeostasis during bone formation.

Preserving and restoring bone with continuous insulin infusion therapy in a mouse model of type 1 diabetes

Those with type 1 diabetes (T1D) are more likely to suffer a fracture than age- and sex-matched individuals without diabetes, despite daily insulin therapy. In rodent studies examining the effect of bone- or glucose-targeting therapies on preventing the T1D-related decrease in bone strength, insulin co-therapy is often not included, despite the known importance of insulin signaling to bone mass accrual. Therefore, working toward a relevant pre-clinical model of diabetic bone disease, we assessed the effect of continuous subcutaneous insulin infusion (CSII) therapy at escalating doses on preserving bone and the effect of delayed CSII on rescuing the T1D-related bone deterioration in an established murine model of T1D. Osmotic minipumps were implanted in male DBA/2J mice 2weeks (prevention study) and 6weeks (rescue study) after the first injection of streptozotocin (STZ) to deliver insulin at 0, 0.0625, 0.125, or 0.25IU/day (prevention study; n=4–5 per dose) and 0 or 0.25IU/day (rescue study; n=10 per group). CSII lasted 4weeks in both studies, which also included age-matched, non-diabetic DBA/2J mice (n=8–12 per study). As the insulin dose increased, blood glucose decreased, body weight increased, a serum maker of bone resorption decreased, and a serum marker of bone formation increased such that each end-point characteristic was linearly correlated with dose. There were insulin dose-dependent relationships (femur diaphysis) with cross-sectional area of cortical bone and cortical thickness (micro-computed tomography) as well as structural strength (peak force endured by the mid-shaft during three-point bending). Likewise, trabecular bone volume fraction (BV/TV), thickness, and number (distal femur metaphysis) increased as the insulin dose increased. Delayed CSII improved glycated hemoglobin (HbA1c), but blood glucose levels remained relatively high (well above non-diabetic levels). Interestingly, it returned the resorption and formation markers to similar levels as those seen in non-T1D control mice. This apparent return after 4weeks of CSII translated to a partial rescue of the structural strength of the femur mid-shaft. Delayed CSII also increased Tb.Th to levels seen in non-T1D controls but did not fully restore BV/TV. The use of exogenous insulin should be considered in pre-clinical studies investigating the effect of T1D on bone as insulin therapy maintains bone structure without necessarily lowering glucose below diabetic levels.

The peripheral quantitative computed tomographic and densitometric analysis of skeletal tissue in male Wistar rats after chromium sulfate treatment.

This study evaluates the effects of three different doses of chromium sulphate on bone density and the tomographic parameters of skeletal tissue of rats. The experiment was performed on 40 male Wistar rats which received, by gavage, during 90 days, a chromium sulphate in either a daily dose of 400, 600 or 800 µg/kg BW. At the end of experiment, the rats were scanned using the densitometry method (DXA) to determine the bone mineral density, bone mineral content of total skeleton and vertebral column (L2-L4) and parameters of body composition (Lean Mass and Fat Mass). The isolated femora were scanned using peripheral a quantitative computed tomography method (pQCT) for a separate analysis of the trabecular and cortical bone tissue. The ultimate strength, work to ultimate and the Young modulus of femora was also investigated by the three-point bending test. The negative impact of chromium was observed in relation to bone tissue. All doses significantly decreased total skeleton density and mineral content, and also had impact upon the isolated femora and vertebral column. Trabecular volumetric bone mineral density and trabecular bone mineral content measured by pQCT in distal femur metaphysis were significantly lower in the experimental groups than in the control. Higher doses of chromium also significantly decreased values of ultimate strength and Young modulus in the investigated femora. The results of the experiment demonstrate that chromium sulphate is dose dependent, and exerts a disadvantageous effect on the skeleton, as it decreases bone density and resistance.

LncRNA-H19 Modulates Wnt/β-catenin Signaling by Targeting Dkk4 in Hindlimb Unloaded Rat.

To investigate the biological functions of long noncoding RNA-H19 (H19) in the pathogenesis of disuse osteoporosis (DOP). Fifty-four male Sprague Dawley (SD) rats were randomly divided into three groups: baseline control (BC, 6), age-matched control (AC, 24), and hindlimb unloading (HLU, 24). The rats in the BC group were sacrificed at the beginning of the experiment, while the AC and HLU rats were sacrificed at different times (7, 14, 21 and 28 days after HLU). The DOP model was verified by micro-CT scan, and quantitative real-time polymerase chain reaction (qRT-PCR) was used to quantify the expression of osteogenic genes (OPG, RunX2 and OPG). Gene sequencing and bioinformatic analysis were performed to find H19 target genes and the associated signaling pathway, which were first verified on tissue samples. Further verification was performed by knocking down the H19 and related gene in rat osteoblast cell line (UMR106 cell). Then, the changes of associated signaling pathway and osteogenic function were examined to confirm the prediction of the bioinformatic analysis. Micro-CT scans and quantitative real-time polymerase chain reaction (qRT-PCR) tests showed progressively deteriorated trabecular bone and decreased level of osteogenic genes in the metaphysis of distal femur during HLU, indicating the successful establishment of a DOP model. According to RNA sequencing, 1351 mRNA and 464 lncRNA were abnormally expressed in response to mechanical unloading, in which the H19 decreased 2.86 fold in HLU rats. There were 1426 mRNA predicted to be the target genes of H19, and KEGG pathway analysis suggested that Wnt signaling pathway (Wnt signaling) was the top pathway responsible for these target genes. In the Wnt-associated genes targeted by H19, 11 were differentially expressed between HLU and AC rats, among which Dkk4 increased 2.44 fold in HLU rats when compared to normal controls. These results of sequencing and bioinformatic analysis were confirmed by the low expression of H19, overexpression of Dkk4 and inhibited Wnt signaling observed in DOP rats. Subsequent in vitro cell assay further demonstrated that knockdown of H19 led to upregulation of Dkk4, and inhibition of Wnt signaling and osteogenic function in UMR106 cell. These effects can be greatly reversed after application of knocking down Dkk4. Our findings demonstrated that low expression of H19, induced by mechanical unloading, leads to development of DOP through inhibition of Wnt signaling by promoting Dkk4 expression.

Disuse osteopenia induced by botulinum toxin is similar in skeletally mature young and aged female C57BL/6J mice

Osteopenia and osteoporosis predominately occur in the fully grown skeleton. However, it is unknown whether disuse osteopenia in skeletally mature, but growing, mice resembles that of fully grown mice. Twenty-four 16-week-old (young) and eighteen 44-week-old (aged) female C57BL/6J mice were investigated. Twelve young and nine aged mice were injected with botulinum toxin in one hind limb; the remaining mice served as controls. The mice were euthanized after 3weeks of disuse. The femora were scanned by micro-computed tomography (µCT) and bone strength was determined by mechanically testing the femoral mid-diaphysis and neck. At the distal femoral metaphysis, the loss of trabecular bone volume fraction (BV/TV) differed between the young and aged mice. However, at the distal femoral epiphysis, no age-dependent differences were observed. Thinning of the trabeculae was not affected by the age of the mice at either the distal femoral metaphysis or the epiphysis. Furthermore, the aged mice lost more bone strength at the femoral mid-diaphysis, but not at the femoral neck, compared to the young mice. In general, the bone loss induced by botulinum toxin did not differ substantially between young and aged mice. Therefore, the loss of bone in young mice resembles that of aged mice, even though they are not fully grown.

Layer-by-Layer Anatomy of the Anterolateral Complex of the Knee

Injuries to the anterior cruciate ligament can be associated with anterolateral capsular injuries. Recently, these capsular tears have been suggested to play an important role in rotatory knee instability. A critical review of the anterolateral complex of the knee is necessary to clarify nomenclatures of same structures and the complex anatomy of anterolateral side of the knee. In addition to the lateral collateral ligament and the arcuate complex, the iliotibial band (ITB) is an important lateral structure. It consists of different layers, namely, the superficial, middle, deep, and capsulo-osseous layers. Further, in a subset of patients, a capsular thickening, that is, the mid-third capsular ligament can be observed. Biomechanically, the functional unit of the ITB with its deeper layers between the firm attachment on the distal lateral femoral metaphysis (Kaplan fibers) and Gerdy tubercle was shown to contribute the most to resist internal tibial rotation. Therefore, the ITB and its deeper layers as well as the mid-third capsular ligament should be referred to collectively as the anterolateral complex.

Stimulation of Osteogenic Differentiation by Saikosaponin-A in Bone Marrow Stromal Cells Via WNT/β-Catenin Pathway.

Saikosaponin-A (SA), a class of native compound with numerous biological activities, may exert protective effect against postmenopausal bone loss. However, it remains unknown whether SA regulates the osteogenic differentiation of bone marrow stromal cells (BMSCs) in the treatment and prevention of osteoporosis. In this study, BMSCs were treated with various concentrations of SA to stimulate osteogenic differentiation over a 14-day period. Additionally, a canonical ovariectomized (OVX) mouse model was used to evaluate the effect of 3-month SA treatment in preventing postmenopausal osteoporosis. In vitro, we found that SA promotes alkaline phosphatase activity/staining and Alizarin red assay, stimulated the expression of osteogenic markers, i.e., runt-related transcription factor 2 (Runx2), osterix, osteopontin, and osteocalcin (OCN) in BMSCs. In vivo, the trabecular number, trabecular thickness, and trabecular bone mineral density of the distal femoral metaphysis were significantly increased in OVX mice treated intraperitoneally with SA for 3months compared with OVX mice that not treated with SA. Moreover, the expression of Runx2 and OCN in OVX + SA mice was significantly increased than that in OVX mice. Finally, we found that SA activated the WNT/β-catenin pathway and the expression of several downstream genes including T-cell factor-1 and lymphoid enhancer factor-1. Inhibition of WNT/β-catenin pathway by Dickkopf-related protein 1 blocked the positive role of SA on osteogenesis. Therefore, SA promoted the osteogenic differentiation of BMSCs through WNT/β-catenin signaling.

Isopsoralen-mediated suppression of bone marrow adiposity and attenuation of the adipogenic commitment of bone marrow-derived mesenchymal stem cells.

Osteoporosis (OP) increases the risk of bone fractures and other complications, and is thus a major clinical problem. In this study, we examined the effect of isopsoralen on the differentiation of bone-derived marrow mesenchymal stem cells (BMSCs) into osteoblasts and adipocytes, as well as bone formation under osteoporotic conditions. Primary femoral BMSCs isolated from C57BL/6 mice were used to evaluate the isopsoralen-mediated regulation of the expression of alkaline phosphatase (ALP), osteocalcin (OCN) and runt-related transcription factor 2 (RUNX2) during osteogenesis 2 weeks. We also examined the expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein β (C/EBPβ) under adipogenic conditions for 1 and 2 weeks. In addition, ovariectomized (OVX) mice were used to examine the effects of isopsoralen on bone formation for 2 months. Finally, mammalian target of rapamycin complex 1 (mTORC1) signaling was examined under osteogenic and adipogenic conditions. We found that following treatment with isopsoralen, the expression levels of ALP, OCN and RUNX2 were upregulated, whereas those of PPARγ and C/EBPβ were downregulated. mTORC1 signaling was also inhibited in vitro and in vivo. In the OVX mice that were intragastrically administered isopsoralen, bone parameters (trabecular thickness, bone volume/total volume and trabecular number) in the distal femoral metaphysis were significantly increased and the adipocyte number was decreased. On the whole, our findings demonstrate that isopsoralen promoted BMSC differentiation into osteoblasts and suppressed differentiation into adipocytes.

Morphological features osteointegration of porous tantalum implants in rats

Among the metallic materials, porous tantalum attracts grow­ing attention of orthopedics and traumatologists. The purpose of the study is to investigate osseointegration by using a porous tantalum implants in rats with normal bone health and osteopo­rosis simulated background. Methods: t he study was performed in 18 laboratory rats weighing 250 – 350 g. Osteoporosis modeled in 9 animals with the help of ovariectomy in 3 months prior to implantation. 3 mm diameter defect produced in the distal femoral metaphysis in normal rats (control) and with osteopo­rosis (experimental), porous tantalum pins implanted. After 14, 30 and 90 days postoperatively the material was examined by using a histology techniques. Re­sults: i t was found that the orientation of osteoreparation pro­cess was identical for both groups of animals, ie at all stages of monitoring, the formation of bone around the implant. However, in comparison with the control group osteoporosis animals had larger areas of direct contact with the tantalum implant intertrabecular spaces of the bone marrow and the formation of the parent bone connective tissue sections. In control and ex­perimental animals 90 days postoperatively bone tissue formed around the implant was presented collagen type I with a different refraction of collagen fibers. It is proved a good biologi­cal compatibility of porous tantalum implants with bone tissue and bone marrow, as well as higher quality of osseointegration. In animals with a normal structure of the bone implant 73.1 % of the perimeter was surrounded by mature bone tissue. In rats with osteoporosis this value was also quite high and amount­ed to 57.7 %. Conclusions: t he study has evidence indicating that tantalum implants can be used in orthopedics and trauma­tology in patients with normal bone health and osteoporosis.

Long-Term Oral Toxicity and Anti-osteoporotic Effect of Sintered Dicalcium Pyrophosphate in Rat Model of Postmenopausal Osteoporosis

Sintered dicalcium pyrophosphate (SDCP), a synthetic pyrophosphate analog, has shown potential for the management of osteoporosis. The long-term oral toxicity and anti-osteoporotic effect of SDCP in a postmenopausal osteoporosis rat model were evaluated in this study. SDCP was orally administered to bilateral ovariectomized (OVX) Wistar rats at a dose of 0.75 mg/kg daily for 24 weeks following by 2 weeks of observation. There were no abnormal findings in clinical signs of toxicity, food consumption, body weight, blood examination, necropsy, and histological inspection attributable to the ingestion of SDCP. The serum level of type I collagen fragments, a bone resorption marker, decreased in SDCP-treated rats, and the bone formation markers alkaline phosphatase, osteocalcin, and osteopontin significantly decreased. These findings indicate that the bone turnover rate decreased in SDCP-treated animals. Relative to OVX rats, the increase in serum tartrate-resistant acid phosphatase 5b level represents an increase in bony tissues in the SDCP-treated rats. Histological examinations of distal femoral metaphyses further revealed that the ingestion of SDCP improved the trabecular bone architecture and decreased bone porosity. Analysis of limb bone ashes showed a significant increase in bone mineral content. Our results show that SDCP inhibits bone resorption to restore bone mass in OVX rats without deleterious effects, and therefore that SDCP has potential in the management of osteoporosis.

Fish Oil with Higher DHA Content and Voluntary Exercise Decreases Postmenopausal Bone Loss

There is increasing evidence suggesting that fish oil (FO) decreases bone resorption by reducing osteoclastogenesis and regular exercise (EX) increases bone mass. EX is associated with increasing bone formation. Therefore, the combined effects of FO intake and EX may have additive effects by both increasing bone formation and decreasing bone resorption. To demonstrate this, we studied the effects of FO and EX on the bone of mice after an ovariectomyinduced bone loss. Twelve months old C57BL/6 female mice were either sham operated or ovariectomized, divided into different dietary and EX group and maintained for 3 months before sacrifice. The distal femoral metaphysis (DFM) showed significantly higher total BMC in both the FO sham sedentary groups. 30/20 ovariectomized sedentary mice had higher total bone mineral content (BMC). EX significantly increased BMC in the control and one of the FO ovariectomized (18/12) groups. The total bone mineral density (BMD) was higher in both the FO ovariectomized mice. EX significantly increased BMD in the control and combination with 18/12 ovariectomized groups. Cortical BMC increased after EX with both FO diets. The DFM showed no significant changes in the trabecular thickness. However, the trabecular number increased in mice fed both FO diet and on EX. Trabecular separation was decreased in the control and 30/20 FO-fed animals but increased in the 18/12 FO-fed mice. In conclusion, in the distal femoral metaphysis, FO-fed mice showed increased BMD. In combination with EX, the 30/20 FO-fed mice showed higher trabecular number and cortical bone mass as well as decreased trabecular separation.

Recombinant Erythropoietin and Darbepoetin Alpha Exert a Similar Dose-Dependent Osteopenic Effect Which May Advocate for "Start Low, Go Slow" Dosing Strategy in Clinical Practice

Background and rationale: Identifying risk factors for bone loss is of utmost importance in the prevention of osteoporotic bone fractures. We and others have recently described the dramatic erythropoietin (EPO)-induced bone loss. Recombinant EPO and its long-acting derivative, darbepoetin alpha (DA), are frequently and interchangeably used in clinical practice for similar indications including diseases (e.g. multiple myeloma) in which bone mass may be compromised already, as a part of the disease process. Here, we examined in a murine model, the dose response to the erythroid stimulating agents (ESA) EPO and DA, in the erythroid and bone compartments.Methods:C57B6/JRcchsd 9-week old female mice were treated for two weeks with either EPO or DA using two dose intensities. EPO and DA were administered subcutaneously thrice or once weekly, respectively, for a total weekly dose of 27IU/mouse or 540IU/mouse, hereafter defined correspondingly as intermediate (ID) and high dose (HD) subgroups. Bone morphometry of the distal femoral metaphysis was assessed using microCT. Flow cytometry was used for immunophenotyping of bone marrow (BM) cells. One-way ANOVA was used to compare variables among the treatment groups.Results: First, we found that ID-DA induced bone loss to a similar extent as ID-EPO as reflected by a significant (p<0.05) reduction in the trabecular bone fraction (BV/TV, -31%), bone mineral density (BMD, -53%) and trabecular number (Tb.N, -17%). HD-EPO and HD-DA resulted in even higher degrees of bone mass reduction: -48% in BV/TV and -57% in Tb.N (p<0.01 for both). Noteworthy, despite modestly superior gain (~10%) in hemoglobin level (21 and 22.5 mg/dl p< 0.01 in HD-EPO and ID-EPO, respectively; mean hemoglobin levels in diluent-treated mice was 16.4), administration of HD-EPO resulted in substantially higher bone loss compared to ID-EPO. This was manifested in ~80% further reduction in BV/TV as well as 2.5-fold further drop in Tb.N as demonstrated by ΔBV/TV and ΔTb.N (i.e. reduction relative to the control) of 48% versus 27% and 57% versus 23%, respectively (p<0.02 for both parameters). By flow cytometry EPO, but not DA, induced a 35% reduction in alkaline phosphatase positive CD11b negative (ALPL+/CD11b-) preosteoblast population in the bone marrow (p<0.01). The percentage of CD115+ BM cells, containing osteoclast precursors, was increased by 26-30% following both EPO and DA treatments (p<0.05 for EPO/DA versus control and non-significant p>0.05 for EPO versus DA groups). In view of the established role of B cells in bone metabolism, we determined the effects of EPO and DA on BM B cell compartment. HD-EPO but not ID-EPO induced a "maturation arrest"-like state in the bone marrow B cell population, increasing the proportion of B220+/CD19-/CD43high/IgM- pre-pro-B two-fold and B220+/CD19+/CD43high/IgM- pro-B cells 2.8-fold (p<0.05) while reducing the proportion of B220+/CD19+/CD43low/IgM+immature B cells by 2.3-fold (p<0.01). Intriguingly, these alterations were not noted following DA treatment.Conclusion:Applying clinically comparable dosing regimen, both recombinant EPO and DA exert a similar, dose-dependent bone loss, not directly proportional to the erythroid response. However, DA, in contrast to EPO, spares the preosteoblast and bone marrow B cell populations. These finding suggest a different mechanism of action of these closely related ESA preparations on the skeletal and immune systems thus warranting further exploration. Finally, our findings advocate for initiating and maintaining patients with the lowest possible doses of EPO/DA to minimize untoward skeletal and other extra-erythropoietic effects, while achieving the desired correction of anemia.This study was supported by a grant from the Israel Cancer Association to DN, YG and MM. DisclosuresNo relevant conflicts of interest to declare.

Effect of osteoporosis on fixation of osseointegrated implants in rats.

The effect of osteoporosis on implant osseointegration has been widely investigated, whereas osteoporosis may also newly occur in patient with previously osseointegrated implant. This study was designed to investigate the effect of osteoporosis on implant fixation in rats after successful osseointegration had been obtained. Seventy female Sprague-Dawley rats were included, and each animal received two titanium implants in the distal metaphysis of femur bilaterally. Eight weeks later, ten rats were sacrificed to confirm the establishment of implant osseointegration. All left rats were randomly subjected to bilateral ovariectomy (OVX) or sham operation. Three, six, and twelve weeks later, implant osseointegration, peri-implant bone tissue, and biomechanical properties of implant were analyzed. Right femurs with implants were used for micro-CT and histological analysis, and left femurs with implants were used for biomechanical test. Micro-CT, histology, and biomechanical test confirmed the destructive effect of OVX on previously osseointegrated implant in rats; when compared to sham-operated rats, peri-implant bone volume, trabecular architecture, bone-to-implant contact ratio, as well as biomechanical parameters decreased progressively within 12 weeks. Results also indicated that the effect of OVX on undisturbed bone (proximal tibiae) was much stronger than that on peri-implant bone. Osteoporosis produced a progressive negative effect on previously osseointegrated implant in distal femora of rats during 12 weeks. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2426-2432, 2017.

Effects of chronic sleep deprivation on bone mass and bone metabolism in rats.

BackgroundThis study aimed to assess the effects of chronic sleep deprivation (CSD) on bone mass and bone metabolism in rats.MethodsTwenty-four rats were randomly divided into CSD and control (CON) groups. Rats were subjected to CSD by using the modified multiple platform method (MMPM) to establish an animal model of CSD. Biochemical parameters such as levels of serum N-terminal propeptide of type I procollagen (PINP), N-terminal cross-linking telopeptide of type I collagen (NTX), growth hormone (GH), estradiol (E2), serum 25(OH)D, and calcium (Ca) were evaluated at 0, 1, 2, and 3 months. After 3 months, each fourth lumbar vertebra and the distal femoral metaphysis of the left extremity of rats were harvested for micro-computed tomography scans and histological analysis, respectively, after the rats were sacrificed under an overdose of pentobarbital sodium.ResultsCompared with rats from the CON group, rats from the CSD group showed significant decreases in bone mineral density (BMD), bone volume over total volume, trabecular bone thickness, and trabecular bone number and significant increases in bone surface area over bone volume and trabecular bone separations (P < 0.05). Bone histomorphology studies showed that rats in the CSD group had decreased osteogenesis, impaired mineralization of newly formed bones, and deteriorative trabecular bone in the secondary spongiosa zone. In addition, they showed significantly decreased levels of serum PINP (1 month later) and NTX (3 months later) (P < 0.05). The serum 25(OH)D level of rats from the CSD group was lower than that of rats from the CON group after 1 month (P < 0.05).ConclusionsCSD markedly affects bone health by decreasing BMD and 25(OH)D, deteriorating the bone microarchitecture, and decreasing bone formation and bone resorption markers.

Efficacy of systemic administration of oxytocin on implant osseointegration in osteoporotic rats

This study investigated the effects of systemic administration of oxytocin (OT) in osteoporotic rats on implant osseointegration. Twenty rats were randomly assigned to the control and experimental groups. Initially, the rats underwent bilateral ovariectomy. After 12 weeks, an osteoporosis model was established. Each rat received an implant at the distal and middle femoral metaphysis. Simultaneously, systemic administration was conducted with one group receiving subcutaneous injection of OT (1 mg·kg⁻¹ per day), whereas the other group received placebo injection. After treatment for 4 weeks, another surgery was conducted to remove the thigh bones from the rats containing the implants for an eight-week observation. With the employment of micro-CT, histological observation and push-out test, osseointegration was evaluated. While the rats received thigh-bone removal surgery, another surgery was conducted to remove the tibia metaphysis from the rats of both groups to perform histological observation and micro-CT inspection. The trabecular bone of tibial samples was intensive and formed woven mesh structure in the experimental group compared with the control group. In the experimental group, the relative bone volume/tissue volume surrounding the implant, the bone contact ratio, and the maximum push-out force of the implant were 0.35%±0.06%, 67.25%±9.06%, and (70.32±10.91) N, respectively, the corresponding values were 0.11%±0.02%, 43.25%±7.01% and (21.65±4.36) N in the control group, and the experimental group increased significantly compared with the control group (P<0.05). Systemic administration of OT cannot only antagonize the negative effects of osteoporosis but can also promote implant healing and osseointegration of pure titanium implants.

Combination Therapy with Zoledronic Acid and Parathyroid Hormone Improves Bone Architecture and Strength following a Clinically-Relevant Dose of Stereotactic Radiation Therapy for the Local Treatment of Canine Osteosarcoma in Athymic Rats.

Clinical studies using definitive-intent stereotactic radiation therapy (SRT) for the local treatment of canine osteosarcoma (OSA) have shown canine patients achieving similar median survival times as the current standard of care (amputation and adjuvant chemotherapy). Despite this, there remains an unacceptable high risk of pathologic fracture following radiation treatment. Zoledronic acid (ZA) and parathyroid hormone (PTH) are therapeutic candidates for decreasing this fracture risk post-irradiation. Due to differing mechanisms, we hypothesized that the combined treatment with ZA and PTH would significantly improve bone healing more than ZA or PTH treatment alone. Using an orthotopic model of canine osteosarcoma in athymic rats, we evaluated bone healing following clinically-relevant doses of radiation therapy (12 Gy x 3 fractions, 36 Gy total). Groups included 36 Gy SRT only, 36 Gy SRT plus ZA, 36 Gy SRT plus ZA and PTH, 36 Gy SRT plus PTH, and 36 Gy SRT plus localized PTH treatment. Our study showed significant increases in bone volume and increased polar moments of inertia (in the distal femoral metaphysis) 8 weeks after radiation in the combined (ZA/PTH) treatment group as compared to radiation treatment alone. Histomorphometric analysis revealed evidence of active mineralization at the study endpoint as well as successful tumor-cell kill across all treatment groups. This work provides further evidence for the expanding potential indications for ZA and PTH therapy, including post-irradiated bone disease due to osteosarcoma.

Room temperature housing results in premature cancellous bone loss in growing female mice: implications for the mouse as a preclinical model for age-related bone loss.

Room temperature housing (22°C) results in premature cancellous bone loss in female mice. The bone loss was prevented by housing mice at thermoneutral temperature (32°C). Thermogenesis differs markedly between mice and humans and mild cold stress induced by standard room temperature housing may introduce an unrecognized confounding variable into preclinical studies. Female mice are often used as preclinical models for osteoporosis but, in contrast to humans, mice exhibit cancellous bone loss during growth. Mice are routinely housed at room temperature (18-23°C), a strategy that exaggerates physiological differences in thermoregulation between mice (obligatory daily heterotherms) and humans (homeotherms). The purpose of this investigation was to assess whether housing female mice at thermoneutral (temperature range where the basal rate of energy production is at equilibrium with heat loss) alters bone growth, turnover and microarchitecture. Growing (4-week-old) female C57BL/6J and C3H/HeJ mice were housed at either 22 or 32°C for up to 18weeks. C57BL/6J mice housed at 22°C experienced a 62% cancellous bone loss from the distal femur metaphysis during the interval from 8 to 18weeks of age and lesser bone loss from the distal femur epiphysis, whereas cancellous and cortical bone mass in 32°C-housed mice were unchanged or increased. The impact of thermoneutral housing on cancellous bone was not limited to C57BL/6J mice as C3H/HeJ mice exhibited a similar skeletal response. The beneficial effects of thermoneutral housing on cancellous bone were associated with decreased Ucp1 gene expression in brown adipose tissue, increased bone marrow adiposity, higher rates of bone formation, higher expression levels of osteogenic genes and locally decreased bone resorption. Housing female mice at 22°C resulted in premature cancellous bone loss. Failure to account for species differences in thermoregulation may seriously confound interpretation of studies utilizing mice as preclinical models for osteoporosis.

Micro-computed tomography assisted distal femur metaphyseal blunt punch compression for determining trabecular bone strength in mice

Shorter generation time and the power of genetic manipulation make mice an ideal model system to study bone biology as well as bone diseases. However their small size presents a challenge to perform strength measurements, particularly of the weight-bearing cancellous bone in the murine long bones. We recently developed an improved method to measure the axial compressive strength of the cancellous bone in the distal femur metaphysis in mice. Transverse micro-computed tomography image slices that are 7µm thick were used to locate the position where the epiphysis–metaphysis transition occurs. This enabled the removal of the distal femur epiphysis at the exact transition point exposing the full extent of metaphyseal trabecular bone, allowing more accurate and consistent measurement of its strength. When applied to a murine model system consisting of five month old male wild-type (WT) and Ca2+/calmodulin dependent protein kinase kinase 2 (CaMKK2) knockout (KO) Camkk2−/− mice that possess recorded differences in trabecular bone volume, data collected using this method showed good correlation between bone volume fraction and strength of trabecular bone. In combination with micro-computed tomography and histology, this method will provide a comprehensive and consistent assessment of the microarchitecture and tissue strength of the cancellous bone in murine mouse models.

Inhibition of bone metastasis by fish oil, thymoquinone, and xanthone

Breast cancer is the second most common form of cancer in the United States of America. If left untreated, breast cancer cells metastasize to the bone. At this stage, osteoclastogenesis is increased and bone loss is accelerated, causing fragile bones. Bone metastasis produces severe bone pain, and currently available medical treatment only focuses on reducing the pain and not on tumor growth or decreasing metastasis. Moreover, medications cause severe side effects, therefore, there is an increase in the use of alternative medicine and nutritional supplementation to decrease bone metastasis. The objectives of this research study, was to determine if fish oils (FO) with high docosahexaenoic acid content by itself or in combination with, thymoquinone (TQ) and xanthones (XN) would decrease bone metastasis in female mice. Four week old C57BL/6 athymic female mice were divided into the following groups: Group 1. Control (C); Group 2. Control Tumor (CT); Group 3. FO fed; Group 4. TQ fed; Group 5. XN fed; Group 6. FO+TQ; Group 7. FO+XN; and Group 8. TQ+XN. Athymic mice were fed their respective diets for 4 or 6 weeks and then groups 2 to 8 were injected with MDA‐MB‐231 breast cancer cells. Feeding their respective diets were continued for an additional 4 weeks. After four weeks, mice were euthanized, femurs and tibias were collected and scanned with a peripheral quantitative computed tomography (pQCT) densitometer.The scans of the right distal femur metaphysis (DFM) were analyzed and both FO and FO+TQ expressed the most significant and consistent bone protective properties, when compared to the CT group. The Total Bone Area (Tot B Ar) significantly decreased in the CT group (10%, p&lt;0.05), when compared to that of the C group, but significantly increased in all the treatment groups. The total bone mineral content (Tot BMC) increased significantly in the FO+TQ treated group (6%, p&lt;0.05). Trabecular bone mineral density (Tb BMD) increased in the FO (10%, p&lt;0.05) and FO+TQ treated groups (14%, p&lt;0.05), in comparison with the CT group. Although the other diets did attenuate bone metastasis in the DFM, they were to a lesser extent compared to the FO and FO+TQ groups.The proximal tibial metaphysis (PTM) showed increased Tot B Ar in the FO (24%, p&lt;0.05), TQ (19%, p&lt;0.05), FO+XN (24%, p&lt;0.05) and TQ+XN (24%, p&lt;0.05. The Trabecular Bone Area (Tb B Ar) also increased significantly in all the different treatments tested, compared to those of the CT mice. The Trabecular Bone Mineral Content (Tb BMC) was increased significantly after treated with FO (31%, p&lt;0.05), TQ (53%, p&lt;0.05) and TQ+XN (44%, p&lt;0.05), when compared to those of the CT mice.In summary, FO protected the Tot B Ar and Tb BMD in the femur, while in the tibia, in addition to the Tot B Ar, it protected the Tb b Ar and Tb BMC. We conclude that FO protected femur and tibia from bone metastasis better than TQ and XN alone.Support or Funding InformationUniversity Research Initiative