Increase of PCSK9 expression in diabetes promotes VEGFR2 ubiquitination to inhibit endothelial function and skin wound healing.

Diabetic foot ulcers (DFUs) are a serious vascular disease. Currently, no effective methods are available for treating DFUs. Pro-protein convertase subtilisin/kexin type 9 (PCSK9) regulates lipid levels to promote atherosclerosis. However, the role of PCSK9 in DFUs remains unclear. In this study, we found that the expression of PCSK9 in endothelial cells (ECs) increased significantly under high glucose (HG) stimulation and in diabetic plasma and vessels. Specifically, PCSK9 promotes the E3 ubiquitin-protein ligase NEDD4 binding to vascular endothelial growth factor receptor 2 (VEGFR2), which led to the ubiquitination of VEGFR2, resulting in its degradation and downregulation in ECs. Furthermore, PCSK9 suppresses the expression and activation of AKT, endothelial nitric oxide synthase (eNOS), and ERK1/2, leading to decreased nitric oxide (NO) production and increased superoxide anion (O2._) generation, which impairs vascular endothelial function and angiogenesis. Importantly, using evolocumab to limit the increase in PCSK9 expression blocked the HG-induced inhibition of NO production and the increase in O2._ production, as well as inhibited the phosphorylation and expression of AKT, eNOS, and ERK1/2. Moreover, evolocumab improved vascular endothelial function and angiogenesis, and promoted wound healing in diabetes. Our findings suggest that targeting PCSK9 is a novel therapeutic approach for treating DFUs.

The oxidized phospholipid PGPC impairs endothelial function by promoting endothelial cell ferroptosis via FABP3.

Ferroptosis is a novel cell death mechanism that is mediated by iron-dependent lipid peroxidation. It may be involved in atherosclerosis development. Products of phospholipid oxidation play a key role in atherosclerosis. 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) is a phospholipid oxidation product present in atherosclerotic lesions. It remains unclear whether PGPC causes atherosclerosis by inducing endothelial cell ferroptosis. In this study, human umbilical vein endothelial cells (HUVECs) were treated with PGPC. Intracellular levels of ferrous iron, lipid peroxidation, superoxide anions (O2•-), and glutathione were detected, and expression of fatty acid binding protein-3 (FABP3), glutathione peroxidase 4 (GPX4), and CD36 were measured. Additionally, the mitochondrial membrane potential (MMP) was determined. Aortas from C57BL6 mice were isolated for vasodilation testing. Results showed that PGPC increased ferrous iron levels, the production of lipid peroxidation and O2•-, and FABP3 expression. However, PGPC inhibited the expression of GPX4 and glutathione production and destroyed normal MMP. These effects were also blocked by ferrostatin-1, an inhibitor of ferroptosis. FABP3 silencing significantly reversed the effect of PGPC. Furthermore, PGPC stimulated CD36 expression. Conversely, CD36 silencing reversed the effects of PGPC, including PGPC-induced FABP3 expression. Importantly, E06, a direct inhibitor of the oxidized 1-palmitoyl-2-arachidonoyl-phosphatidylcholine IgM natural antibody, inhibited the effects of PGPC. Finally, PGPC impaired endothelium-dependent vasodilation, ferrostatin-1 or FABP3 inhibitors inhibited this impairment. Our data demonstrate that PGPC impairs endothelial function by inducing endothelial cell ferroptosis through the CD36 receptor to increase FABP3 expression. Our findings provide new insights into the mechanisms of atherosclerosis and a therapeutic target for atherosclerosis.

High-density lipoprotein regulates angiogenesis by long non-coding RNA HDRACA.

Normal high-density lipoprotein (nHDL) can induce angiogenesis in healthy individuals. However, HDL from patients with coronary artery disease undergoes various modifications, becomes dysfunctional (dHDL), and loses its ability to promote angiogenesis. Here, we identified a long non-coding RNA, HDRACA, that is involved in the regulation of angiogenesis by HDL. In this study, we showed that nHDL downregulates the expression of HDRACA in endothelial cells by activating WW domain-containing E3 ubiquitin protein ligase 2, which catalyzes the ubiquitination and subsequent degradation of its transcription factor, Kruppel-like factor 5, via sphingosine 1-phosphate (S1P) receptor 1. In contrast, dHDL with lower levels of S1P than nHDL were much less effective in decreasing the expression of HDRACA. HDRACA was able to bind to Ras-interacting protein 1 (RAIN) to hinder the interaction between RAIN and vigilin, which led to an increase in the binding between the vigilin protein and proliferating cell nuclear antigen (PCNA) mRNA, resulting in a decrease in the expression of PCNA and inhibition of angiogenesis. The expression of human HDRACA in a hindlimb ischemia mouse model inhibited the recovery of angiogenesis. Taken together, these findings suggest that HDRACA is involved in the HDL regulation of angiogenesis, which nHDL inhibits the expression of HDRACA to induce angiogenesis, and that dHDL is much less effective in inhibiting HDRACA expression, which provides an explanation for the decreased ability of dHDL to stimulate angiogenesis.

Closing-opening wedge osteotomy for the treatment of congenital kyphosis in children.

BACKGROUND: To evaluate the safety and effectiveness of posterior closed-open wedge osteotomy for treatment of congenital kyphosis in children. METHODS: Imaging and clinical data from January 2010 to December 2019 of posterior closed-open wedge osteotomy of congenital kyphosis with at least 2-year follow up was analyzed retrospectively. Perioperative indicators such as operation time, osteotomy site, osteotomy method and occurrence of complications, and imaging indicators were observed. The 3D printed models were used to measure the expanded distance of anterior edge vertebra and closed length of spinal canal line. The clinical effect was evaluated through SRS-22 questionnaires. RESULTS: There were 15 CK patients in this study. The osteotomy segments and details are as follows: 1 case each for T6-9 and L2, 2 cases at T11, 3 cases at T12, and 6 cases at L1. The average operation time was 314 min, the average blood loss was 970 mL, the average fusion range was 6.3 segments, and the average time of follow up was 70.5 months. The Cobb angle of local kyphosis was corrected from 65.6 ± 18.8° to 11.3 ± 7.1°(p < .001). The range of kyphosis correction was 40-90°, and average correction rate was 83.2% (67.7-95.7%). The correction was stable in follow-up, and the kyphotic angle was 11.0 ± 7.6 (p = .68). The preoperative SVA was 31.5 ± 21.8 mm, and the postoperative recovery was 18.0 ± 15.5, while the last follow-up was 9.1 ± 7.9. The p values were 0.02 and 0.07 respectively. By using 3D printed models, the expanded distance of anterior edge vertebra and closed length of spinal canal line were 14.5 ± 7.5 mm and 24.5 ± 8.0 mm respectively. Self-image and satisfaction in SRS-22 improved significantly. There was no recurrence of deformity and junctional kyphosis. CONCLUSIONS: The posterior closing-opening wedge osteotom for treatment of congenital kyphosis in children is satisfactory, if selected appropriately. During the longitudinal follow-up, the patients could achieve solid fusion and the correction could be well maintained.Evidence of Confidence: IVa.

What Is the Best Way for Patients to Take Photographs of Medical Images (Radiographs, CT, and MRI) Using a Smartphone?

BACKGROUND: Teleradiology has become one of the most important approaches to virtual clinical diagnosis; its importance has only grown during the coronavirus 2019 pandemic. In developing countries, asking patients to take photographs of their images using a smartphone can facilitate the process and help keep its costs down. However, the images taken by patients with smartphones often are of poor quality, and there is no regulation or standard instruction about how to use smartphones to take photographs of medical examination images effectively. These problems limit the use of smartphones in remote diagnosis and treatment. QUESTIONS/PURPOSES: To formulate a set of guidelines for the most appropriate and effective use of smartphones to capture images (radiographs, CT images, and MR images), and to determine whether these guidelines are more effectively adopted by patients of differing ages and genders. METHODS: In this prospective study, a set of step-by-step instructions was created with the goal of helping patients take better smartphone photographs of orthopaedic diagnostic images for transfer to telemedicine services. Following the advice of surgeons, experts in smartphone technology, imaging experts, and suggestions from patients, the instructions were modified based on clinical experience and finalized with the goals of simplicity, clarity, and convenience. Potentially eligible patients were older than 18 years, had no cognitive impairment, and used smart phones. Based on that, 256 participants (patients or their relatives and friends) who visited the orthopaedic department of our hospital from June to October 2020 potentially qualified for this study. A total of 11% (29) declined to participate, leaving 89% (227) for analysis here. Their mean age was 36 ± 11 years, 50% were women (113 of 227), and the patient himself/herself represented in 34% (78 of 227) of participants while relatives or friends of patients made up 66% (149 of 227) of the group. In this study, the diagnoses included spinal stenosis (47% [107 of 227]), disc herniation without spinal stenosis (31% [71 of 227]), vertebral fractures (14% [32 of 227]), and other (7% [17 of 227]). Each study participant first took photographs of their original medical images based on their own knowledge of how to use the smartphone camera function; each participant then took pictures of their original images again after receiving our instructional guidance. Three senior spine surgeons (YZ, TQL, TCM) in our hospital analyzed, in a blinded manner, the instructed and uninstructed imaging files based on image clarity (the content of the image is complete, the text information in the image is clearly visible, there is neither reflection nor shadow in the image) and image position (it is not tilted, curled, inverted, or reversed). If either of these conditions was not satisfied, the picture quality was deemed unacceptable; two of three judges' votes determined the outcome. Interobserver reliability with kappa values for the three judges were 0.89 (YZ versus TQL), 0.92 (YZ versus TCM), and 0.90 (TQL versus TCM). RESULTS: In this study, the overall proportion of smartphone medical images deemed satisfactory increased from 40% (91 of 227) for uninstructed participants to 86% (196 of 227) for instructed participants (risk ratio 2.15 [95% CI 1.82 to 2.55]; p<0.001). The proportion of acceptable-quality images in different age groups improved after instruction, except for in patients aged 51 years or older (3 of 17 uninstructed participants versus 8 of 17 instructed participants; RR 2.67 [95% CI 0.85 to 8.37]; p = 0.07). The proportion of acceptable-quality images in both genders improved after instruction, but there was no difference between the genders. CONCLUSION: We believe our guidelines for patients who wish to take smartphone photographs of their medical images will decrease image transmission cost and facilitate orthopaedic telemedicine consultations. However, it appears that patients older than 50 years are more likely to have difficulty with this approach, and if so, they may benefit from more hands-on assistance from clinic staff or younger relatives or friends. The degree to which our findings are culture-specific should be verified by other studies in other settings, but on the face of it, there is little reason to believe our findings would not generalize to a reasonable degree. Other studies in more heterogeneous populations should also evaluate factors related to levels of educational attainment and wealth differences, but in the meantime, our findings can give clinical teams an idea of which patients may need a little extra assistance. LEVEL OF EVIDENCE: Level II, therapeutic study.

Evaluation of Anterior Decompression Surgical Outcomes of Proximal-Type Cervical Spondylotic Amyotrophy: A Retrospective Study.

OBJECTIVE: To investigate the efficiency of anterior decompression on the proximal-type cervical spondylotic amyotrophy patients. METHODS: This was a retrospective analysis. From January 2014 to November 2017, 21 patients with proximal-type cervical spondylotic amyotrophy (CSA) underwent anterior decompression. There were 15 males and 6 females, aged 35-73 years with an average of 51.62 years. All the patients underwent surgery of anterior decompression (ACDF or ACCF). Among them, 12 patients underwent C4/5 single level ACDF, eight patients underwent C4/5 and C5/6 double level ACDF, and one patient underwent C5 anterior cervical corpectomy decompression and fusion surgery. Preoperative and postoperative clinical and radiologic parameters were assessed. The clinical examinations were reviewed, including muscle strength, neck disability index (NDI) score, cervical Japanese Orthopaedic Association (JOA) score, and improvement rate of manual muscle test (MMT) at the last follow-up. Preoperative spinal cord or nerve impingement was assessed by magnetic resonance imaging (MRI) or computed tomography (CT) myelography. Postoperative lateral X-ray radiographs were performed every 3 months after the surgery. RESULTS: Severe preoperative muscle atrophy of the deltoid or biceps muscles occurred in 21 patients included in the study. All of them involve impingements of the ventral nerve root and/or the anterior horn according to MRI and CT myelography. The preoperative duration of symptoms averaged 8.4 months. The average follow-up for all patients was 13.2 months. At the final follow-up, all patients showed statistically significant improvements in muscle strength and NDI scores (P < 0.05, P < 0.05). For the deltoid muscles force and C-JOA scores, the average improvement rates were 66.49% ± 10.04% and 62.23% ± 9.23%, respectively. With respect to MMT, 12 proximal-type patients were graded excellent, six were good, and three were fair, and the overall improvement rate was 85.7%. CONCLUSIONS: For proximal-type CSA patients with cervical radiculopathy, earlier anterior decompression surgery can achieve satisfactory results by significantly improving a patient's muscle strength and relieving compression symptoms.

Therapeutic effect of minimally invasive catheter drainage and local chemotherapy for the lumbosacral tuberculosis without neural symptoms.

Increasing number of studies have shown growing incidence of lumbosacral tuberculosis and its complications. However, the treatment options for this disorder are still limited.To evaluate the long-term therapeutic effect and prognosis of minimally invasive puncture catheter drainage and Isoniazid local chemotherapy for the treatment of lumbosacral tuberculosis without neural symptoms under the guidance of computed tomography (CT).A total of 45 patients with asymptomatic lumbosacral tuberculosis were treated by minimally invasive catheter drainage under CT guidance. Forty-two cases had been followed up, which included 22 women and 20 men with an average age of 36.45 years old. Isoniazid was injected locally and antituberculotic drugs were administered for postoperative treatment. Oswestry Disability Index (ODI), visual analogue scale (VAS) evaluation and Cobb angle were recorded before and after operation.Forty-two patients had been followed up and the follow-up term was from 1.2 to 8.5 years (average 60 months). All patients were healed without recurrent cases. The ODI were improved from 14.86 ±â€Š2.02 before operation to 1.48 ±â€Š1.55 after operation. The post-operative (4.19 ±â€Š1.17) VAS score was improved compared to the pre-operative VAS score (0.55 ±â€Š0.55). The post-operative Cobb angle (6.19°â€Š±â€Š3.85°) was also improved relatively to the preoperative Cobb angle (5.90°â€Š±â€Š3.71°).Minimally invasive puncture catheter drainage combined with Isoniazid local chemotherapy is an effective method for lumbosacral tuberculosis without neural symptom. Meanwhile, it can be applied for the treatment of spinal tuberculosis before open surgery.

A new method for calculating the desired laminoplasty opening size based on the target sagittal canal diameter before single-door cervical laminoplasty.

PURPOSE: To build a mathematical model which could calculate the desired laminoplasty opening size (LOS) based on the target sagittal canal diameter (SCD) before single-door cervical laminoplasty (SDCL) when taking the effects of surgery drill into consideration. METHODS: The model was based on geometric analysis on deformation of spinal canal; the formula was derived and characterized as: y (mm) = 2 [Formula: see text] × sin(ß/2) = c - d (y is the size of LOS, [Formula: see text] the size of transverse canal diameter, ß the size of laminoplasty opening size, c the size of mini-plate and d the diameter of the drill bit used during the surgery operation). The parameters of pre- and postoperative computed tomography scans of 20 patients who had undergone SDCL were measured by the picture archiving and communication system (PACS) software and a new instrument named as Lei's ruler, respectively. RESULTS: The effects of surgery SDCL were very significant; for each patient, the SCD was enlarged dramatically after the surgery (P < 0.01). The differences between the data obtained by PACS and Lei's ruler were no statistically significant (P > 0.05). According to the derived formula, the 95% confidence intervals of SCD after the surgery were within the range of 14 mm and 14.5 mm. CONCLUSION: Applying the mathematical model and derived formula, the desired LOS could be calculated according to the target SCD which could help the surgeon select an optimum mini-plate before SDCL. At the same time, a new measuring device named Lei's ruler is designed for the convenience of the derived formula. These slides can be retrieved under Electronic Supplementary Material.

Magnesium Ions Promote the Biological Behaviour of Rat Calvarial Osteoblasts by Activating the PI3K/Akt Signalling Pathway.

Magnesium has been investigated as a biodegradable metallic material. Increased concentrations of Mg2+ around magnesium implants due to biodegradation contribute to its satisfactory osteogenic capacity. However, the mechanisms underlying this process remain elusive. We propose that activation of the PI3K/Akt signalling pathway plays a role in the Mg2+-enhanced biological behaviours of osteoblasts. To test this hypothesis, 6, 10 and 18 mM Mg2+ was used to evaluate the stimulatory effect of Mg2+ on osteogenesis, which was assessed by evaluating cell adhesion, cell viability, ALP activity, extracellular matrix mineralisation and RT-PCR. The expression of p-Akt was also determined by western blotting. The results showed that 6 and 10 mM Mg2+ elicited the highest stimulatory effect on cell adhesion, cell viability and osteogenic differentiation as evidenced by cytoskeletal staining, MTT assay results, ALP activity, extracellular matrix mineralisation and expression of osteogenic differentiation-related genes. In contrast, 18 mM Mg2+ had an inhibitory effect on the behaviour of osteoblasts. Furthermore, 10 mM Mg2+ significantly increased the phosphorylation of Akt in osteoblasts. Notably, the aforementioned beneficial effects produced by 10 mM Mg2+ were abolished by blocking the PI3K/Akt signalling pathway through the addition of wortmannin. In conclusion, these results demonstrate that 6 mM and 10 mM Mg2+ can enhance the behaviour of osteoblasts, which is at least partially attributed to activation of the PI3K/Akt signalling pathway. Furthermore, a high concentration (18 mM Mg2+) showed an inhibitory effect on the biological behaviour of osteoblasts. These findings advance the understanding of cellular responses to biodegradable metallic materials and may attract greater clinical interest in magnesium.

A novel total cervical prosthesis for single-level cervical subtotal corpectomy: radiologic and histomorphometric analysis in a caprine model.

STUDY DESIGN: A novel total cervical prosthesis (TCP) for single-level cervical subtotal corpectomy was assessed in a caprine animal model. OBJECTIVES: To investigate the radiologic and histomorphometric characteristics of a novel TCP for single-level cervical subtotal corpectomy. SUMMARY OF BACKGROUND: Cervical disk replacement has emerged as a promising alternative to arthrodesis in the management of cervical disk herniation. However, they are designed for anterior cervical discectomy, and not suitable for cervical subtotal corpectomy. To solve this problem, our group has developed a novel TCP for single-level cervical subtotal corpectomy. MATERIALS AND METHODS: There were 12 adult Shannxi goats (2 y old) used in this study. The goats were divided into 2 groups based on postoperative survival periods of 3 (n=6) and 6 (n=6) months after surgery. Using an anterior surgical approach, a standard anterior C3 vertebra subtotal corpectomy and decompression of the spinal canal were performed, followed by implantation of the TCP device. Then all the goats were killed and underwent radiographic and histologic observations. RESULTS: The TCP implant procedures were successfully completed in all 12 goats without incidence of vascular or infectious complications. The range of motion of C2-C3 and C3-C4 segments were preserved in both of the groups. Three-dimensional images of specimens interface indicated confluent interdigitization of trabeculae at the prosthetic endplate-bone interface, without evidence of significant radiolucent lines or gaps. Histomorphometric analysis showed that there were a large number of fibrous tissue and a small amount of cartilage cells between the prostheses and bone in the 3 months' group. In the 6 months' group, part of fibrous tissue has changed into the cartilage tissue. CONCLUSIONS: Our data show that this prosthesis can maintain the stability of the cervical spine and retain the activity of the cervical spine in vivo. The findings in this study provide a foundation for ongoing clinical investigations using the TCP.

Osseointegration of chitosan coated porous titanium alloy implant by reactive oxygen species-mediated activation of the PI3K/AKT pathway under diabetic conditions.

Chitosan coated porous titanium alloy implant (CTI) is demonstrated a promising approach to improve osseointegration capacity of pure porous titanium alloy implant (TI). Since chitosan has been demonstrated to exhibit antioxidant activity, we propose CTI may ameliorate the ROS overproduction, thus reverse the poor osseointegration under diabetic conditions, and investigate the underlying mechanisms. Primary rat osteoblasts incubated on the TI and the CTI were subjected to normal serum (NS), diabetic serum (DS), DS + NAC (a potent ROS inhibitor) and DS + LY294002 (a PI3K/AKT-specific inhibitor). In vivo study was performed on diabetic sheep implanted with TI or CTI into the bone defects on crista iliaca. Results showed that diabetes-induced ROS overproduction led to osteoblast dysfunction and apoptosis, concomitant with the inhibition of AKT in osteoblasts on the TI substrate. While CTI stimulated AKT phosphorylation through ROS attenuation, thus reversed osteoblast dysfunction evidenced by improved osteoblast adhesion, increased proliferation and ALP activity, and decreased cytotoxicity and apoptotic rate, which exerted same effect to NAC treatment on the TI. These effects were further confirmed by the improved osseointegration within the CTI in vivo evidenced by Micro-CT and histological examinations. In addition, the aforementioned promotive effects afforded by CTI were abolished by blocking PI3K/AKT pathway with addition of LY294002. These results demonstrate that the chitosan coating markedly ameliorates diabetes-induced impaired bio-performance of TI via ROS-mediated reactivation of PI3K/AKT pathway, which elicits a new surface functionalization strategy for better clinical performance of titanium implant in diabetic patients.

What is the work environment of orthopaedic surgeons in China?

BACKGROUND: Physicians in China face heavy demands from patients and the government for services but deal with the threat of unpredictable legal and physical conflicts with patients, some ending with the death of doctors. More than 40 doctors and nurses have been killed by patients since 2001. QUESTIONS/PURPOSES: We sought to evaluate (1) the demographics of orthopaedic practice, (2) duty periods, (3) practice support, and (4) job satisfaction among orthopaedic surgeons in China. METHODS: Questionnaires were posted online at www.OrthoChina.org for download by orthopaedic surgeons in 2006 to 2007, and sent to those attending meetings in 2013. In 2013, a total of 1350 surgeons were invited and 456 participated in the survey at meetings. In 2007, during the period of the survey, 9759 individuals were qualified orthopaedic surgeons, and 334 participated in the survey at www.OrthoChina.org . RESULTS: Ninety-one percent of orthopaedic surgeons work in public and 9% in private hospitals. Ninety-four percent work more than 8 hours per day 6 to 7 days a week. Twenty-five percent work more than 12 hours per day 6 to 7 days a week without extra compensation. The majority of orthopaedic surgeons must work on national statutory holidays. Almost none received contractually mandated income for weekends and national holidays. Approximately 80% of participants reported an attack of some kind, including physical or psychologic harm. With respect to job satisfaction, 73% stated they would not choose to be a physician again and 86% reported that they do not want their children to become a physician. CONCLUSIONS: China's rapid economic growth and resulting demands for modern health care have resulted in heavy pressure on orthopaedic surgeons, financially and personally. Chinese orthopaedic surgeons are overworked, suffer lack of respect, and face the possibility of serious personal harm. As a consequence, they are demoralized and unsatisfied. Significant reforms are needed.

The promotion of osteointegration under diabetic conditions using chitosan/hydroxyapatite composite coating on porous titanium surfaces.

Composited Chitosan/Hydroxyapatite (CS/HA) material coated on titanium surface (cTi) is a promising approach to produce biomaterials with better osseointegration capacity, but its bio-performance under diabetic conditions and the mechanisms involved remain elusive. We propose that the alterations in the Wnt/ß-catenin pathway may play a role in mediating the improvement effect of cTi on diabetes-induced impaired implant osteointegration. To confirm the hypothesis, primary rat osteoblasts incubated on Ti and cTi were subjected to normal serum (NS), diabetic serum (DS), DS + Wnt3a (a specific Wnt agonist) and DS + Dkk1 (a specific Wnt antagonist) treatment. In vivo study was performed on diabetic sheep implanted with Ti or cTi into the bone defect on crista iliaca. Results showed that diabetes depressed osteoblast function evidenced by impaired cell adhesion and morphology, decreased cell proliferation and ALP activity, and higher apoptotic rate on Ti. Importantly, both cTi and Wnt3a treatment ameliorated osteoblastic dysfunction and apoptosis under diabetic condition. Implantation with cTi significantly improved osteointegration evidenced by Micro-CT and histological examinations compared with Ti. Moreover, the aforementioned promotive effects afforded by cTi were abolished by blocking Wnt pathway with Dkk1. Our study explicitly demonstrates that CS/HA composite material improves diabetes-induced impaired osteointegration of Ti via the reactivation of Wnt/ß-catenin pathway and provides a target point for biomaterial modification to attain better clinical performance in diabetic patients.

Repair of segmental bone defect using Totally Vitalized tissue engineered bone graft by a combined perfusion seeding and culture system.

BACKGROUND: The basic strategy to construct tissue engineered bone graft (TEBG) is to combine osteoblastic cells with three dimensional (3D) scaffold. Based on this strategy, we proposed the "Totally Vitalized TEBG" (TV-TEBG) which was characterized by abundant and homogenously distributed cells with enhanced cell proliferation and differentiation and further investigated its biological performance in repairing segmental bone defect. METHODS: In this study, we constructed the TV-TEBG with the combination of customized flow perfusion seeding/culture system and ß-tricalcium phosphate (ß-TCP) scaffold fabricated by Rapid Prototyping (RP) technique. We systemically compared three kinds of TEBG constructed by perfusion seeding and perfusion culture (PSPC) method, static seeding and perfusion culture (SSPC) method, and static seeding and static culture (SSSC) method for their in vitro performance and bone defect healing efficacy with a rabbit model. RESULTS: Our study has demonstrated that TEBG constructed by PSPC method exhibited better biological properties with higher daily D-glucose consumption, increased cell proliferation and differentiation, and better cell distribution, indicating the successful construction of TV-TEBG. After implanted into rabbit radius defects for 12 weeks, PSPC group exerted higher X-ray score close to autograft, much greater mechanical property evidenced by the biomechanical testing and significantly higher new bone formation as shown by histological analysis compared with the other two groups, and eventually obtained favorable healing efficacy of the segmental bone defect that was the closest to autograft transplantation. CONCLUSION: This study demonstrated the feasibility of TV-TEBG construction with combination of perfusion seeding, perfusion culture and RP technique which exerted excellent biological properties. The application of TV-TEBG may become a preferred candidate for segmental bone defect repair in orthopedic and maxillofacial fields.

[Three-dimensional flow perfusion culture enhances proliferation of human fetal osteoblasts in large scaffold with controlled architecture].

OBJECTIVE: To demonstrate the feasibility and benefits of custom designed perfusion bioreactor in conjunction with well-defined three-dimensional (3D) environment for enhanced proliferation and homogeneous distribution of human fetal osteoblasts in large scaffold in vitro. METHODS: Large-scale ß-tricalcium phosphate (ß-TCP) scaffolds with tightly controlled architectures were fabricated. And a custom designed perfusion bioreactor was developed. Human fetal osteoblasts were seeded onto the scaffolds, cultured for up to 16 days in static or flow perfusion conditions. At Days 4, 8 & 16 post-incubation, the proliferation and distribution of osteoblasts were determined by daily D-glucose consumption, cell viability (methyl thiazolyl tetrazolium (MTT) assay), histological evaluation and scanning electron microcopy (SEM). Sphere like structures observed in the SEM images were assessed by energy dispersive X-ray (EDX) analysis. RESULTS: In both static and perfusion cultures, the daily D-glucose consumption increased with prolonged time. The daily D-glucose consumption was significantly higher in the perfusion culture than that in static culture (P < 0.05). The increased cell viability with time during the culture was similar to the daily D-glucose consumption under both conditions. There was much greater cell viability under flow perfusion culture compared to static culture (P < 0.05). Flow perfused constructs demonstrated improved cell proliferation and a homogeneous layer composed of cells and extracellular matrix in channels throughout the whole scaffold. However, the cells were biased to periphery in scaffolds culture statically. Sphere like structures present in the matrix were identified as calcium phosphate nodules via EDX analysis. CONCLUSIONS: Flow perfusion culture plus well-defined 3D interconnected channel environments enhances the proliferation and improve the distribution of human fetal osteoblasts in large scaffolds. Scaffolds with controlled architecture may be a potential tool of studying the fluid flow configuration and cell behavior inside scaffold in details. And human fetal osteoblasts can be used as a cell source in large bone graft research.

Clinical assessment of a CMC/PEO gel to inhibit postoperative epidural adhesion formation after lumbar discectomy: a randomized, control study.

PURPOSE: To evaluate effectiveness of carboxymethylcellulose/polyethylene oxide (CMC/PEO) gel in improving clinical outcomes after the first-time lumbar discectomy. METHOD: Ninety-three patients with herniated lumbar disc at L4-L5 or L5-S1 were enrolled and randomized into two groups: CMC/PEO gel treatment group and control group. All the patients underwent laminotomy and discectomy by posterior approach. The preoperative and postoperative Oswestry Disability Index (ODI) and Visual Analogue Scale (VAS) scores for lower-back pain and leg pain were analyzed and compared between two groups at 30- and 60-day time points. RESULTS: No patient presented with any clinically measurable adverse event during surgery. There were no significant differences between the treated group and the control group on the preoperative ODI and VAS scores. In general, the ODI and VAS scores decreased in both groups at all the time points. At the 30-day time point, the VAS scores for back pain and leg pain and the ODI scores in treatment group were lower by 9.9 % (P = 0.0302), 27.0 % (P = 0.0002) and 16.3 % (P = 0.0007) than those in control group. And at the 60-day time point, the ODI and VAS scores further decreased in both groups. The VAS scores for leg pain in treatment group were lower by 4.5 % than that in the control group (P = 0.0149). However, no significant difference was detected between two groups on the ODI and VAS scores for back pain. CONCLUSIONS: The results demonstrated that CMC/PEO gel is effective in reducing posterior dural adhesions in the spine with no apparent safety issues. It can improve patients' postoperative clinical outcome.

Effect of reactive oxygen species overproduction on osteogenesis of porous titanium implant in the present of diabetes mellitus.

Clinical evidence indicates diabetes as a majorrisk factor for titaniumimplant treatment with high failure rates and poor osteointegration, but the underlying mechanism involved remains elusive.We hypothesize that reactive oxygen species (ROS) overproduction may contribute to the impaired osteogenesis of porous titanium implants (pTi) under diabetic conditions. To test this hypothesis, we culturedprimary rabbit osteoblasts onto pTi and studied the cellular performance when subjected to normal serum (NS), diabetic serum (DS), DS + NAC (a potent ROS inhibitor) and NS + H(2)O(2)(an oxidant).In-vivo performance of pTi was investigated by transplanting them intofemoral condyledefects of diabetic rabbits, which received vehicle or NAC treatment respectively.Results showed that diabetic conditions induced significant cellular apoptosis, depressedosteoblast function evidenced by impairedcell attachment and morphology, decreased cell proliferation anddifferentiation, andcompromised in-vivo osteogenesis ofpTi, while cellular ROSgeneration was increased derived from mitochondrial dysfunction. Scavenging ROS with NAC markedly attenuated cell apoptosis and osteoblast dysfunction, and improved bone ingrowth within pTi. Furthermore, treatment withH(2)O(2) exerted similar adverse effect on cellular behavior as diabetes. This study furthers our knowledge on the potential role of ROS overproduction in the diabetes-induced impaired osteogenesis of titanium implants, and indicates anti-oxidative treatment as a promising strategy to promote the treatment efficacy of pTi in diabetic patients.

Influence of architecture of ß-tricalcium phosphate scaffolds on biological performance in repairing segmental bone defects.

BACKGROUND: Although three-dimensional (3D) ß-tricalcium phosphate (ß-TCP) scaffolds serve as promising bone graft substitutes for the segmental bone defect treatment, no consensus has been achieved regarding their optimal 3D architecture. METHODS: In this study, we has systematically compared four types of ß-TCP bone graft substitutes with different 3D architectures, including two types of porous scaffolds, one type of tubular scaffolds and one type of solid scaffolds, for their efficacy in treating segmental bone defect in a rabbit model. RESULTS: Our study has demonstrated that when compared to the traditional porous and solid scaffolds, tubular scaffolds promoted significantly higher amount of new bone formation in the defect regions as shown by X-ray, micro CT examinations and histological analysis, restored much greater mechanical properties of the damaged bone evidenced by the biomechanical testing, and eventually achieved the complete union of segmental defect. Moreover, the implantation of tubular scaffolds enhanced the neo-vascularization at the defect region with higher bone metabolic activities than others, as indicated by the bone scintigraphy assay. CONCLUSIONS: This study has further the current knowledge regarding the profound influence of overall 3D architecture of ß-TCP scaffolds on their in vivo defect healing performance and illuminated the promising potential use of tubular scaffolds as effective bone graft substitute in treating large segmental bone defects.