By adopting the method of voxel-mirrored homotopic connectivity (VMHC), the correlation of the synchrony of spontaneous neural functional activities between symmetrical regions is determined to be mediated by the corpus callosum (CC). This study investigated differences in homotopic functional connectivity (FC) across distinct symptom spread directions in individuals with upper motor neuron-dominant amyotrophic lateral sclerosis (UMN-D ALS). The UMN-D ALS patients were categorized into two groups on the basis of the direction of symptom spread—horizontal spread (group H) and vertical spread (group V). Indices of interhemispheric functional and structural changes are derived via analyses of VMHC and probabilistic fiber tracking. The VMHC analysis of grey matter revealed that the intergroup differences in the superior frontal gyrus (SFG) were greater in group H than in groups V and HC (voxel P < 0.05, cluster P < 0.05, GRF corrected). According to CC-based VMHC analysis, group H had greater VMHC values than did group V (45 mm³ vs. 18 mm³ at a voxel-level threshold of P < 0.05, uncorrected). In UMN-D ALS, the results of VMHC analysis vary with different spread directions. In group H, homotopic FC significantly increased, possibly associated with early bilateral limb involvement and subsequent compensatory increases in the SFG. These results contribute to the understanding of the relationship between brain function and symptom evolution in individuals with ALS.

Background Bone formation requires coordinated communication between vascular and osteogenic compartments. Although simvastatin has been reported to stimulate both vascular growth and osteoblast activity, the molecular process by which these responses are integrated during bone anabolism remains incompletely defined. This study explored whether VEGF serves as a functional link between simvastatin-induced vascular remodeling and osteogenic activation. Methods Sixty male Sprague–Dawley rats were randomly assigned into five groups: normal control, vehicle control, intraosseous simvastatin administration (0.5 mg), intraperitoneal bevacizumab administration, and combined intraosseous simvastatin plus intraperitoneal bevacizumab treatment. Skeletal mass, trabecular architecture, dynamic bone formation, and local vascular networks were evaluated using DXA, μCT, histomorphometry, and Microfil® perfusion. Circulating osteogenic and angiogenic factors were measured by ELISA, and protein-level changes were assessed by Western blotting. In parallel, MC3T3-E1 osteoblast-lineage cells were used to examine alkaline phosphatase activity, BMP-2 production, BMP-2 secretion, and endoplasmic reticulum localization. Results Intraosseous simvastatin substantially improved bone mineral density, trabecular microstructure, and mineral apposition, together with increased serum VEGF and osteocalcin levels. These skeletal effects were accompanied by expansion of intraosseous and periosteal vascular networks. VEGF blockade largely attenuated these simvastatin-associated anabolic and vascular responses. In MC3T3-E1 cells, bevacizumab reduced osteoblast ALP activity, was associated with altered BMP-2 processing and reduced extracellular release, and increased the overlap between BMP-2 and the ER marker calnexin. These findings support a VEGF-dependent contribution to BMP-2 processing and secretion, while not establishing VEGF as the sole regulator of this pathway. Conclusions Simvastatin promotes bone formation by coordinating vascular expansion with osteoblast functional maturation through a VEGF-dependent component. The data are consistent with a role for VEGF signaling in BMP-2 processing and secretion, linking angiogenic activation to osteogenic output while requiring further studies to define the direct molecular mechanism.

INTRODUCTION:Neuromuscular ultrasound has been increasingly used in the detection and diagnosis of amyotrophic lateral sclerosis (ALS), commonly characterized by peripheral nerve atrophy, degeneration, and muscle fasciculations. The aim of this study was to assess the ultrasound characteristics of ALS patients. METHODS:A total of 67 consecutive patients with sporadic ALS and 19 with ALS mimics (63.16% peripheral neuropathy) were recruited. Ultrasound and electrophysiological examinations were performed; the peripheral nerve cross-sectional area (CSA) and fasciculation grades were compared between the groups, and correlations between ultrasound and electrophysiological data in ALS patients were determined. RESULTS:ALS patients had smaller proximal median and ulnar nerve CSAs than those of ALS mimics, who exhibited asymmetric changes. Fasciculation differences in the trapezius, triceps brachii, extensor digitorum communis, thenar, and first dorsal interosseous muscles were observed. In ALS patients, the CSA and fasciculation relative scores were correlated with electrophysiological indicators. CONCLUSION:Ultrasound is a valuable tool for monitoring peripheral nerve CSA and muscle fasciculations, both of which correlate with electrophysiological indices, in ALS patients.

INTRODUCTION:Mirror movements (MMs) are often overlooked in patients with amyotrophic lateral sclerosis (ALS). Although the contralateral co-movement (COMO) test can be used to evaluate MMs in patients with ALS, it lacks a systematic evaluation. The aim of this study was to validate the effectiveness of the Chinese version of the COMO test in a Chinese ALS population. METHODS:We prospectively enrolled 173 patients with ALS as the disease group and 28 healthy individuals as controls. All participants were evaluated using the Chinese version of the COMO test. Univariate analysis and multiple linear regression were used to compare differences between groups. Subgroup analysis of the COMO scores was performed based on different disease characteristics. RESULTS:The COMO score in the ALS group was significantly greater (5.00% [1.67-10.00]) than that in the healthy control group (1.67% [0.00-3.33]). After adjusting for confounders, this difference remained significant. Multivariate linear analysis suggested that the upper motor neuron (UMN) score independently predicted the COMO score (P < 0.001). The COMO score was not affected by different onset regions or lateralizations. Propensity score matching revealed no significant difference in COMO scores between uninvolved limb segments and the corresponding limb segments in other patients. The Cronbach's α of the Chinese COMO test was 0.621. CONCLUSION:The Chinese COMO test can serve as a potential tool for assessing MMs in Chinese patients with ALS. The UMN score is a factor influencing the COMO score. The COMO test can provide objective evidence for ALS characteristics and the severity of UMN damage.

Objective Damage to the corpus callosum (CC) in amyotrophic lateral sclerosis (ALS) patients has been confirmed via electrophysiological, neuroimaging, and autopsy studies. Additionally, the CC is hypothesized to serve as a pathway for the spread of pathological information. This study aimed to investigate whether the CC plays a mediating role in the symptomatic spread of ALS.Methods In this observational study, diffusion tensor imaging (DTI) data were acquired from 45 individuals with the upper motor neuron-dominant (UMN-D) phenotype of ALS. The UMN-D ALS patients were categorized into two groups based on the direction of symptom spread, including 25 patients with horizontal spread (group H) and 20 patients with vertical spread (group V). Diffusivity indices were derived through whole-brain analysis and probabilistic fiber tracking.Results According to the voxel-based analysis and tract-based spatial statistics, differences in axial diffusivity (AD) in the CC were observed between disease subgroups, with patients in group H showing higher AD values than those in group V. Fiber tracking analysis revealed persistent differences in the AD indices of CC-primary motor cortex (PMC) fibers between the two disease subgroups.Conclusion In UMN-D ALS patients, the direction of symptom spread may be related to the degree of CC involvement. The AD metric may be a more specific indicator of CC damage.

Amyotrophic lateral sclerosis is a rare neurodegenerative disease characterized by the involvement of both upper and lower motor neurons. Early bilateral limb involvement significantly affects patients' daily lives and may lead them to be confined to bed. However, the effect of upper and lower motor neuron impairment and other risk factors on bilateral limb involvement is unclear. To address this issue, we retrospectively collected data from 586 amyotrophic lateral sclerosis patients with limb onset diagnosed at Peking University Third Hospital between January 2020 and May 2022. A univariate analysis revealed no significant differences in the time intervals of spread in different directions between individuals with upper motor neuron-dominant amyotrophic lateral sclerosis and those with classic amyotrophic lateral sclerosis. We used causal directed acyclic graphs for risk factor determination and Cox proportional hazards models to investigate the association between the duration of bilateral limb involvement and clinical baseline characteristics in amyotrophic lateral sclerosis patients. Multiple factor analyses revealed that higher upper motor neuron scores(hazard ratio [HR] = 1.05, 95% confidence interval [CI] = 1.01–1.09, P = 0.018), onset in the left limb(HR = 0.72, 95% CI = 0.58–0.89, P = 0.002), and a horizontal pattern of progression(HR = 0.46, 95% CI = 0.37–0.58, P < 0.001) were risk factors for a shorter interval until bilateral limb involvement. The results demonstrated that a greater degree of upper motor neuron involvement might cause contralateral limb involvement to progress more quickly in limb-onset amyotrophic lateral sclerosis patients. These findings may improve the management of amyotrophic lateral sclerosis patients with limb onset and the prediction of patient prognosis.

Postmenopausal osteoporosis is a common bone metabolic disease, and gut microbiota (GM) imbalance plays an important role in the development of metabolic bone disease. Here, we show that ovariectomized mice had high levels of lipopolysaccharide in serum and gut microbiota dysbiosis through increases in luminal Firmicutes:Bacteroidetes ratio. We depleted the GM through antibiotic treatment and observed improvements in bone mass, bone microstructure, and bone strength in ovariectomized mice. Conversely, transplantation of GM adapted to ovariectomy induced bone loss. However, GM depletion reversed ovariectomy-induced gene expression in the tibia and increased periosteal bone formation. Furthermore, bioinformatics analysis revealed that the G-protein-coupled bile acid receptor (TGR5) and systemic inflammatory factors play key roles in bone metabolism. Silencing TGR5 expression through small interfering RNA (siRNA) in the local tibia and knockout of TGR5 attenuated the effects of GM depletion in ovariectomized mice, confirming these findings. Thus, this study highlights the critical role of the GM in inducing bone loss in ovariectomized mice and suggests that targeting TGR5 within the GM may have therapeutic potential for postmenopausal osteoporosis.

Purpose: Chronic heart failure causes osteoporosis, but the mechanism remains unclear. The sympathetic nerve plays an important role in both bone metabolism and cardiovascular function.Methods: Thirty-six adult male SD rats were randomly divided into the following four groups: sham surgery (Sham) group, guanethidine (GD) group, abdominal transverse aorta coarctation-induced heart failure + normal saline (TAC) group, and TAC + guanethidine (TAC + GD) group. Normal saline (0.9 % NaCl) or guanethidine (40 mg/kg/ml) was intraperitoneally injected daily for 5 weeks. Then, DXA, micro-CT, ELISA and RT-PCR an-alyses were performed 12 weeks after treatment.Results: The bone loss in rats subjected to TAC-induced chronic heart failure and chemical sympathectomy with guanethidine was increased. Serum norepinephrine levels were increased in rats with TAC-induced heart failure but were decreased in TAC-induced heart failure rats treated with guanethidine. The expression of alpha 2A adren-ergic receptor, alpha 2C adrenergic receptor, osteoprotegerin (OPG), and osteocalcin in the tibia decreased in the TAC-induced heart failure group, and the expression of beta 1 adrenergic receptor, beta 2 adrenergic receptor, receptor activator of nuclear factor-Kappa B ligand (RANKL), and RANKL/OPG in the tibia increased in the heart failure group. In addition, these changes in gene expression levels were rescued by chemical sympathectomy with guanethidine.Conclusions: TAC-induced chronic heart failure is associated with bone mass loss, and the sympathetic nerve plays a significant role in heart failure-related bone mass loss. Mini abstract: The present study supports the hypothesis that heart failure is related to bone loss, and the excessive activation of sympathetic nerves participates in this pathophysiological process. The present study suggests a potential pathological mechanism of osteoporosis associated with heart failure and new perspectives for developing strategies for heart failure-related bone loss.

Objective: To investigate whether vestibular-evoked myogenic potentials(VEMPs) can be used to assess brainstem involvement in patients with Kennedy's disease (KD). Method: This was a case-control study.Twenty consecutive patients with genetically confirmed KD and 20 age-and sex-matched healthy subjects were enrolled from November 2018 to September 2020.All subjects were tested for three types of VEMPs, including cervical VEMP (c-VEMP) recorded by the sternocleidomastoid muscle (parameter:p13, n23), masseter VEMP (m-VEMP) recorded by the masseter muscle(parameter: p11), and ocular VEMP (o-VEMP) recorded by the inferior oblique muscle (parameter n10, p15).The latency of each wave, interside peak latency and interpeak latency of c-VEMP, the corrected amplitude and amplitude asymmetry ratio were recorded. Bilateral sternocleidomastoid muscle (SCMM) electromyography (EMG) was performed. The spinal cord and bulbous muscular atrophy functional rating scale (SBMAFRS) was used for assessment. Results: The mean p13 latency of c-VEMP was (15.5±1.4)ms, which was longer than that of the control group[(13.3±0.9)ms](P<0.05); the mean n23 latency was(25.5±1.4)ms, which was also longer than that of the control group[(22.5±1.0)ms] (P<0.05); the difference of bilateral p13[(2.3±0.6)ms] was significantly higher than that of the control group(P<0.05). The abnormal rates of c-, m-, o-VEMP in KD patients were 75%(15/20), 30%(6/20) and 20%(4/20), respectively. There was a significant positive correlation between c-VEMP latency and course of disease in KD patients(left: r=0.715, 0.695, right: r= 0.708, 0.715, both P<0.05). However, c-VEMP latency was negatively correlated with SBMAFRS score (left: r=-0.701, -0.694, right: r=-0.644, -0.685, both P<0.05). Abnormal rates of SCMM EMG in KD group were as follows: 15%(3/20)of patients showed spontaneous potential in resting state and 45% (9/20) of patients exhibited simple recruitment. Conclusions: The c-VEMP latency is a sensitive tool for detecting lower brainstem involvement in patients with KD, and the degree of damage increases with prolongation of disease course. The o-and m-VEMP abnormalities indicate that some KD patients develop upper brainstem damage.

Amyotrophic lateral sclerosis (ALS) is one of the fatal neurodegenerative diseases. Muscle ultrasound can be used in ALS to make early diagnosis, strengthen disease management and differentiate other neuromuscular diseases from it. In ALS patients, morphological changes such as muscle atrophy, increased echo intensity and fasciculation can be detected by muscle ultrasound which is helpful in assessing respiratory and swallowing functions as well. High frequency ultrasound has the clinical value in the diagnosis, monitoring and prognosis evaluation of ALS patients.

Objective: We investigated upper motor neuron (UMN) signs in the cervical region in a Chinese clinic-based cohort of patients with flail arm syndrome (FAS) by clinical examination and neurophysiological tests such as triple stimulation technique (TST) and pectoralis tendon reflex testing. Methods: A total of 130 consecutive FAS patients from Peking University Third Hospital underwent physical examination and neurophysiological tests at baseline and 3 months, 6 months, 9 months, and 12 months later. Pyramidal signs, pectoralis tendon reflex and TST results were evaluated to estimate the function of cervical spinal UMNs. Results: At the first visit, weakness of the bilateral proximal upper limbs was found in 99 patients, while weakness of a single proximal upper limb was found in 31 patients. There were 49 patients with tendon hyperreflexia, 42 patients with tendon hyporeflexia and 39 patients with tendon areflexia. All except 4 of the patients had brisk pectoralis tendon reflex. The UMN score of the cervical region was 1.7 ± 0.4, and the lower motor neuron score of that region was 3.5 ± 0.3. The TST test /TST control amplitude ratio was 65.7 ± 7.5%. The latency of quantitative detection of the pectoralis tendon reflex was 7.7 ± 1.2 ms. In the follow-up study, the UMN score and the TST test /TST control amplitude ratio decreased, while the lower motor neuron score increased, and the latency of quantitative detection of the pectoralis tendon reflex remained steady. Conclusion: Although the signs of cervical spinal UMN dysfunction in patients with FAS were often concealed by muscle atrophy in the progression of the disease, TST and pectoralis tendon reflex could reveal it.

OBJECTIVE:To explore the association between rare HSPB1 variants and amyotrophic lateral sclerosis (ALS).METHODS:We performed next-generation sequencing for 166 Chinese ALS patients to screen for possible pathogenic rare variants of HSPB1. The control individuals were obtained from 1000 Genome Project and an in-house whole-exome sequencing database. The Sequence Kernel Association Test (SKAT) and the SKAT-optimal test (SKAT-O) were used to identify the association between rare HSPB1 variants and ALS.RESULTS:We identified 3 possible pathogenic rare variants of HSPB1 (all were missenses), including c.379C>T (p.R127W), c.446A>C (p.D149A) and c.451A>C (p.T151P). Compared with 1000 Genome Project, SKAT p=3.61×10-7 and SKAT-O p=1.62×10-6; while compared with the in-house database, SKAT p=9.99×10-4, SKAT-O p= 1.80×10-3. We analyzed the phenotypes of rare HSPB1 variant carriers and found no specific clinical characteristics associated with these variants.CONCLUSIONS:Rare variants of HSPB1 are probably associated with the pathogenesis of ALS.

The aim of this study was to evaluate the usefulness of the motor unit number index (MUNIX) technique in Kennedy disease (KD) and test the correlation between the MUNIX and other clinical parameters. The MUNIX values of the bilateral deltoid, abductor digiti minimi (ADM), quadriceps femoris (QF), and tibialis anterior (TA) were determined and compared with the course of the disease. The MUNIX sum score was calculated by adding the MUNIX values of these 8 muscles. Disability was evaluated using the spinal and bulbar muscular atrophy functional rating scale (SBMAFRS). The MUNIX scores of patients with KD were negatively correlated with the course of the disease (p < 0.05), whereas their motor unit size index (MUSIX) scores were positively correlated with the course the of disease (p < 0.05). MUNIX sum scores were correlated with SBMAFRS scores (r = 0.714, p < 0.05). MUNIX was more sensitive than compound muscle action potentials or muscle strength as an indicator of neuron loss and axonal collateral reinnervation. The MUNIX sum score is an objective and a reliable indicator of disease progression, and it is a potential choice for therapeutic clinical trials. The MUNIX can assess the functional loss of motor axons and is correlated with disability. The MUNIX sum score may be especially suitable as an objective parameter.

Background: Endothelial progenitor cells have shown the ability to enhance neovascularization. In this study, the authors tested whether intraosseous delivery of simvastatin could mobilize endothelial progenitor cells and enhance recovery in a hindlimb ischemia model. Methods: There are eight groups of rats in this study: normal control; type 1 diabetes mellitus control group control without drug intervention; and type 1 diabetes mellitus rats that randomly received intraosseous simvastatin (0, 0.5, or 1 mg) or oral simvastatin administration (0, 20, or 400 mg). All type 1 diabetes mellitus rats had induced hindlimb ischemia. The number of endothelial progenitor cells in peripheral blood, and serum markers, were detected. The recovery of blood flow at 21 days after treatment was used as the main outcome. Results: The authors demonstrated that endothelial progenitor cell mobilization was increased in the simvastatin 0.5- and 1-mg groups compared with the type 1 diabetes mellitus control and simvastatin 0-mg groups at 1, 2, and 3 weeks. Serum vascular endothelial growth factor levels were significantly increased at 2 weeks in the simvastatin 0.5- and 1-mg groups, in addition to the increase of the blood flow and the gastrocnemius weight at 3 weeks. Similar increase can also been seen in simvastatin 400 mg orally but not in simvastatin 20 mg orally. Conclusion: These findings demonstrate that a single intraosseous administration of simvastatin mobilized endothelial progenitor cells at a dose one-hundredth of the required daily oral dose in rats, and this potent mobilization of endothelial progenitor cells markedly improved diabetic limb ischemia by means of neovascularization.

Objective: To explore the association between rare UBQLN2 variants and amyotrophic lateral sclerosis (ALS) in Chinese population, and the characteristic of phenotypes of their carriers. Methods: A total of 166 ALS patients who visited Department of Neurology of Peking University Third Hospital between January 2018 and July 2020 were recruited. The next-generation sequencing was performed to screen possible pathogenic rare variants of UBQLN2. Meanwhile, control individuals were obtained from 1000 Genome Project (2 504 samples) and an in-house whole-exome sequencing database (1 812 samples), separately. The sequence kernel association test (SKAT) and the SKAT-optimal test (SKAT-O) were used to identify the association between UBQLN2 rare variants and ALS. The clinical characteristics of rare variant carriers were analyzed. Results: A total of 33 familiar ALS and 133 sporadic ALS of Chinese ancestry were enrolled. Of the 166 ALS patients, 12.7% had bulbar-onset, 85.5% had limb-onset, and 5 cases were ALS with frontotemporal dementia (3.0%). The male-to-female ratio was 1.68∶1, with a mean age at symptom onset of (43.8±12.2) years. Three possible pathogenic rare variants of UBQLN2 were detected, including c.128A>G (p.Lys43Arg), c.142G>T (p.Val48Leu) and c.1451T>G (p.Val484Gly), and all of them were novel missense mutations. Compared with 1000 Genome Project, SKAT and SKAT-O showed a P value of 2.49×10-6 and 9.22×10-7, respectively. While compared with the in-house database, SKAT and SKAT-O revealed a P value of 1.42×10-3 and 1.10×10-3, respectively. Patients who carried rare UBQLN2 variants were with a higher rate of bulbar-onset (2/3 vs 19/163, P=0.042). Conclusion: Rare variants of UBQLN2 are associated with ALS in Chinese population, and mutation of UBQLN2 may be relevant to bulbar-onset.

Caloric restriction (CR) is well described and has received extensive attention for its multiple benefits, including longevity and stress resistance. However, some studies have shown that CR negatively influences bone, although a mechanism hasn't been provided. Adiponectin, an adipocyte‐derived hormone, can affect bone metabolism by various pathways. To explore the role of adiponectin in short‐term CR on bone, we tested the effect of short‐term CR on limb bones (tibia and femur) and lumbar vertebral bodies of young C57BL/6 wild‐type (WT) and adiponectin‐deficient (Apn–/–) mice. Two dietary regimes, ad libitum (AL) and CR (70% of the AL diet), were used. Dietary restriction led to increased serum adiponectin in WT mice, while bone mineral density, bone microarchitecture, and biomechanical outcomes of limb bone and vertebrae were decreased. In contrast, bone length, microarchitecture, and biomechanical outcomes were not impaired after CR in Apn–/– mice. Furthermore, CR increased adiponectin expression both in white adipose tissue and bone marrow adipose tissue in young WT mice. Histology analysis showed that expansion of bone marrow adipose tissue after CR in Apn–/– mice was impaired compared with WT mice. These results suggest that increased adiponectin induced by short‐term CR may negatively influence bones.

患者男,50岁,因“肌肉颤动3年半,双上肢无力2年,肌萎缩1年半”于2019年1月17日入院.3年半前家属偶然发现患者胸肌有肌肉颤动,无不适.2年前患者举重物后突发双上肢酸痛无力,并出现上肢肌肉和腹肌负重后痉挛.此后无力及负重后痉挛症状持续存在,均于活动后加重.1个月后患者发现双上肢及胸腹部出现持续性肌肉颤动,不受控制,紧张劳累后加重,睡眠后无缓解.当时外院上肢电生理检查示:右侧臂丛神经传导未见异常,右侧肱二头肌、三角肌、冈下肌神经源性损害.曾口服舒乐安定无效.1年半前患者发现双上肢肌萎缩,右侧为著,肌肉颤动程度逐渐减轻,但范围扩大至双下肢,小腿活动后易痉挛.伴有无力加重,逐渐出现右上肢不能上抬,半年前外院诊为右侧肩袖损伤并手术,术后右上肢痉挛好转,但双上肢无力仍逐渐加重.患者自发病以来,双下肢肌力正常,出汗明显增多.既往血脂异常5年,空腹血糖异常1年;右侧肩袖修补术后半年.吸烟20年,40支/d,已戒烟6年.无特殊接触史.无家族史.

Background: Currently, drugs for local bone formation are very limited in clinic. Simvastatin is one of most potential translational drugs by repositioning for its significant osteogenic effect and its history as a safe lipid-lowering medicine. In this study, we prepared the pre-clinical simvastatin microcrystals which could be potentially translated and industrialized. Methods: Simvastatin microcrystals were made by wet media milling method. Characterization of microcrystal particles were evaluated by laser particle size analyzer and high-performance liquid chromatography (HPLC). Furthermore, we verified the osteogenic effect in rat ovariectomy(OVX)-induced osteoporosis model and femur defect model. For osteoporosis model, we delivered simvastatin microcrystals to tibia with poloxamer hydrogel by intraosseous injection. Bone mineral density (BMD) and ultimate force were assessed after treatment. For femur defect model, simvastatin microcrystal was incorporated in clinically used calcium phosphate cements (CPCs) as an implant. Histology and μCT were used for evaluation of the healing. Results: The D 10 , D 50, and D 90 showed size of the most particles ranged from 0.226-3.425 μm. The mean particle size of the microcrystals is determined as 1.365 μm which demonstrated a successful preparation. For osteoporosis model, BMD and the ultimate force of the treated tibia were significantly improved in osteoporosis rat after injection of 0.5 mg and 1 mg simvastatin microcrystals compared with OVX or 0 mg groups. There were no differences observed in BMD and ultimate force between 0.5 mg, 1 mg simvastatin microcrystals group and bone morphogenetic protein (BMP) 5 ug group. For femur defect model, quantitative analysis of bone regeneration by μCT showed bone volume/tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) were significantly increased in simvastatin microcrystals 50ug (SIM-C 50 ug), SIM-C 100 ug and BMP 5 ug group compared with CPC vehicle group. Trabecular separation (Tb.Sp), and cement volume/tissue volume (CV/TV) were decreased in SIM-C 50 ug, SIM-C 100 ug and BMP 5 ug groups compared with CPC vehicle group. Semiquantitative scale for histology assessment further demonstrated a higher bone regeneration score in drug loaded groups compared with CPC group.Conclusions: Our study shows that simvastatin microcrystals were successfully prepared by wet media milling method and the microcrystals can promote bone formation by local delivery by poloxamer hydrogel or CPC, which is of great translational potential.

Simvastatin is a translational drug that may be used to induce local bone formation. In this study, simvastatin microcrystals were made by a wet media milling method, and then we verified the osteogenic effect of the microcrystals in rat ovariectomy (OVX)-induced osteoporosis and femur defect models. For the osteoporosis model, we delivered simvastatin microcrystals to the tibia with poloxamer hydrogels via an intraosseous injection. Bone mineral density and the ultimate force of the treated tibia were significantly improved after injection of simvastatin microcrystals at 0.5 and 1 mg compared with the OVX or 0-mg control groups. For the femur defect model, simvastatin microcrystals were incorporated in clinically used calcium phosphate cements (CPCs) as an implant. Quantitative analysis of bone regeneration by microcomputed tomography (μCT) showed improved bone morphology with simvastatin microcrystals at 50 and 100 μg, compared with the CPC vehicle. A semiquantitative scale for histology assessment further demonstrated a higher bone regeneration score in the drug-loaded groups. Our study shows that simvastatin microcrystals can promote bone formation by local delivery using a poloxamer hydrogel or CPC, which may be translationally useful.