Progress in the application of graphene and its derivatives to osteogenesis.

As bone and joint injuries from various causes become increasingly prominent, how to effectively reconstruct and repair bone defects presents a difficult problem for clinicians and researchers. In recent years, graphene and its derivatives have been the subject of growing body of research and have been found to promote the proliferation and osteogenic differentiation of stem cells. This provides a new idea for solving the clinical problem of bone defects. However, as as numerous articles address various aspects and have not been fully systematized, there is an urgent need to classify and summarize them. In this paper, for the first time, the effects of graphene and its derivatives on stem cells in solution, in 2D and 3D structures and in vivo and their possible mechanisms are reviewed, and the cytotoxic effects of graphene and its derivatives were summarized and analyzed. The toxicity of graphene and its derivatives is further reviewed. In addition, we suggest possible future development directions of graphene and its derivatives in bone tissue engineering applications to provide a reference for further clinical application.

Enhancement of antibacterial properties and biocompatibility of Ti6Al4V by graphene oxide/strontium nanocomposite electrodepositing.

Ti6Al4V is a widely used orthopedic implant material in clinics. Due to its poor antibacterial properties, surface modification is required to prevent peri-implantation infection. However, chemical linkers used for surface modification have generally been reported to have detrimental effects on cell growth. In this work, by optimizing parameters related to electrodeposition, a composite structural coating with graphene oxide (GO) compact films in the inner layer and 35 nm diameter strontium (Sr) nanoparticles in the outer layer was constructed on the surface of Ti6Al4V without using substance harmful to bone marrow mesenchymal stem cells (BMSCs) growth. The antibacterial properties of Ti6Al4V are enhanced by the controlled release of Sr ions and incomplete masking of the GO surface, showing excellent antibacterial activity against Staphylococcus aureus in bacterial culture assays. The biomimetic GO/Sr coating has a reduced roughness of the implant surface and a water contact angle of 44.1°, improving the adhesion, proliferation and differentiation of BMSCs. Observations of synovial tissue and fluid in the joint in an implantation model of rabbit knee also point to the superior anti-infective properties of the novel GO/Sr coating. In summary, the novel GO/Sr nanocomposite coating on the surface of Ti6Al4V effectively prevents surface colonization of Staphylococcus aureus and eliminates local infections in vitro and in vivo.

Coating CoCrMo Alloy with Graphene Oxide and ε-Poly-L-Lysine Enhances Its Antibacterial and Antibiofilm Properties.

INTRODUCTION: With increases in implant infections, the search for antibacterial and biofilm coatings has become a new interest for orthopaedists and dentists. In recent years, graphene oxide (GO) has been extensively studied for its superior antibacterial properties. However, most of these studies have focused on solutions and there are few antibacterial studies on metal surfaces, especially the surfaces of cobalt-chromium-molybdenum (CoCrMo) alloys. ε-Poly-L-lysine (ε-PLL), as a novel food preservative, has a spectrum of antimicrobial activity; however, its antimicrobial activity after coating an implant surface is not clear. METHODS: In this study, for the first time, a two-step electrodeposition method was used to coat GO and ε-PLL on the surface of a CoCrMo alloy. Its antibacterial and antibiofilm properties against S. aureus and E. coli were then studied. RESULTS: The results show that the formation of bacteria and biofilms on the coating surface was significantly inhibited, GO and ε-PLL composite coatings had the best antibacterial and antibiofilm effects, followed by ε-PLL and GO coatings. In terms of classification, the coatings are anti-adhesive and contact-killing/inhibitory surfaces. In addition to oxidative stress, physical damage to GO and electrostatic osmosis of ε-PLL are the main antibacterial and antibiofilm mechanisms. DISCUSSION: This is the first study that GO and ε-PLL coatings were successfully prepared on the surface of CoCrMo alloy by electrodeposition. It provides a promising new approach to the problem of implant infection in orthopedics and stomatology.

Antibacterial and antibiofilm properties of graphene and its derivatives.

Infections resulting from bacteria and biofilms have become a huge problem threatening human health. In recent years, the antibacterial and antibiofilm effects of graphene and its derivatives have been extensively studied. However, there continues to be some controversy over whether graphene and its derivatives can resist infection and biofilms. Moreover, the antibacterial mechanism and cytotoxicity of graphene and its derivatives are unclear. In the present review, antibacterial and antibiofilm abilities of graphene and its derivatives in solution, on the surface are reviewed, and their toxicity and possible mechanisms are also reviewed. Furthermore, we propose possible future development directions for graphene and its derivatives in antibacterial and antibiofilm applications.

Enhancement of surface bioactivity on carbon fiber-reinforced polyether ether ketone via graphene modification.

BACKGROUND AND OBJECTIVE: The modulus of carbon fiber-reinforced polyether ether ketone (CFR-PEEK), a composite containing layers of carbon fiber sheets, can be precisely controlled to match bone. However, CFR-PEEK is biologically inert and cannot promote bone apposition. The objective of this study was to investigate whether graphene modification could enhance the bioactivity of CFR-PEEK. METHODS AND RESULTS: In vitro, the proliferation and differentiation of rat bone marrow stromal cells on scaffolds were quantified via cell-counting kit-8 assay and Western blotting analysis of osteoblast-specific proteins. Graphene modification significantly promoted bone marrow stromal cell proliferation and accelerated induced differentiation into osteogenic lineages compared to cells seeded onto nongraphene-coated CFR-PEEK. An in vivo rabbit extraarticular graft-to-bone healing model was established. At 4, 8, and 12 weeks after surgery, microcomputed tomography analyses and histological observations revealed significantly better microstructural parameters and higher average mineral apposition rates for graphene-modified CFR-PEEK implants than CFR-PEEK implants (P<0.05). van Gieson staining indicated more new bone was formed around graphene-modified CFR-PEEK implants than CFR-PEEK implants. CONCLUSION: Graphene may have considerable potential to enhance the bioactivity and osseointegration of CFR-PEEK implants for clinical applications.

Graphene coating on the surface of CoCrMo alloy enhances the adhesion and proliferation of bone marrow mesenchymal stem cells.

The objective was to investigate whether a graphene coating could improve the surface bioactivity of a cobalt-chromium-molybdenum-based alloy (CoCrMo). Graphene was produced by chemical vapor deposition and transferred to the surface of the CoCrMo alloy using an improved wet transfer approach. The morphology of the samples was observed, and the adhesion force and stabilization of graphene coating were analyzed by a nanoscratch test and ultrasonication test. In an in vitro study, the adhesion and proliferation of bone marrow mesenchymal stem cells (BMSCs) cultured on the samples were quantified via an Alamar Blue assay and cell counting kit-8 (CCK-8) assay. The results showed that it is feasible to apply graphene to modify the surface of a CoCrMo alloy, and the enhancement of the adhesion and proliferation of BMSCs was also shown in the present study. In conclusion, graphene exhibits considerable potential for enhancing the surface bioactivity of CoCrMo alloy.

Evaluation of the osteogenesis and osseointegration of titanium alloys coated with graphene: an in vivo study.

The aim of this study was to investigate whether a surface coating with graphene could enhance the surface bioactivation of titanium alloys (Ti6Al4V) to further accelerate in vivo osteogenesis and osseointegration at the implant surface. In this study, a New Zealand white rabbit femoral condyle defect model was established. After 4, 12 and 24 weeks, biomechanical testing, micro-computed tomography (Micro-CT) analyses and histological observations were performed. At the highest push-out forces during the test, microstructure parameters, such as the bone volume/total volume fraction (BV/TV) and mineral apposition rate (MAR), of the new bone were significantly higher in the graphene-coated Ti6Al4V group (G-Ti6Al4V) than in the Ti6Al4V group (P < 0.05). Van Gieson (VG) staining showed that the G-Ti6Al4V group had more new bone formation than the Ti6Al4V group, and the G-Ti6Al4V group showed a closer fit between the bone and implant. In conclusion, graphene might be a novel type of nano-coating material for enhancing the surface biological activity of Ti-based alloy materials and may further promote in vivo osteogenesis and osseointegration.

Graphene modified titanium alloy promote the adhesion, proliferation and osteogenic differentiation of bone marrow stromal cells.

We studied the effects of graphene coating on improving the biological activity of a titanium alloy (Ti6Al4V) widely used in hip and knee joint replacements. The experiments included immunofluorescence staining for observing cellular adhesion, Cell Counting Kit-8 (CCK-8) for evaluating cellular proliferation and reverse transcription-polymerase chain reaction (RT-PCR) for detecting the differentiation of bone marrow stromal cells on different scaffolds. The results showed that G-Ti6Al4V exhibited a higher mean integrated optical density (IOD) for vinculin and resulted in a higher cell proliferation rate and higher osteoblast-specific gene transcription levels. In summary, graphene could be used as a new nanocoating material for Ti6Al4V scaffolds to enhance their surface bioactivity.

Effects of Artificial Ligaments with Different Porous Structures on the Migration of BMSCs.

Polyethylene terephthalate- (PET-) based artificial ligaments (PET-ALs) are commonly used in anterior cruciate ligament (ACL) reconstruction surgery. The effects of different porous structures on the migration of bone marrow mesenchymal stem cells (BMSCs) on artificial ligaments and the underlying mechanisms are unclear. In this study, a cell migration model was utilized to observe the migration of BMSCs on PET-ALs with different porous structures. A rabbit extra-articular graft-to-bone healing model was applied to investigate the in vivo effects of four types of PET-ALs, and a mechanical test and histological observation were performed at 4 weeks and 12 weeks. The BMSC migration area of the 5A group was significantly larger than that of the other three groups. The migration of BMSCs in the 5A group was abolished by blocking the RhoA/ROCK signaling pathway with Y27632. The in vivo study demonstrated that implantation of 5A significantly improved osseointegration. Our study explicitly demonstrates that the migration ability of BMSCs can be regulated by varying the porous structures of the artificial ligaments and suggests that this regulation is related to the RhoA/ROCK signaling pathway. Artificial ligaments prepared using a proper knitting method and line density may exhibit improved biocompatibility and clinical performance.

Effects of graphene modification on the bioactivation of polyethylene-terephthalate-based artificial ligaments.

The objective of this study was to investigate whether surface coating with graphene could enhance the surface bioactivation of PET-based artificial ligaments to accelerate graft-to-bone healing after anterior cruciate ligament reconstruction. In an in vitro study, the proliferation of MC3T3-E1 cells and their differentiation on the scaffolds were quantified via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and real-time polymerase chain reaction assays. The significantly higher optical-density values and transcription levels of osteoblast-specific genes indicated that graphene modification could promote the proliferation of MC3T3-E1 cells and accelerate their specific differentiation into osteogenic lineages on scaffolds. In an in vivo test, rabbits were used to establish an extra-articular graft-to-bone healing model. At 4, 8, and 12 weeks after surgery, biomechanical tests, microcomputed tomography analysis, and histological observations were performed. The final results demonstrated that the microstructural parameters, the average mineral apposition rate of the bone, and the biomechanical properties of the graphene-coated polyethylene terephthalate (PET)-based artificial ligament (G-PET-AL) group were significantly higher than those of the PET-AL graft group (P < 0.05). The results of Van Gieson staining indicated that in the G-PET-AL group, there was more newly formed bone than there was in the group in which nongraphene-coated PET-ALs were used. In conclusion, graphene exhibits considerable potential for enhancing the surface bioactivation of materials.

Effects of dimethylolpropionic acid modification on the characteristics of polyethylene terephthalate fibers.

Polyethylene terephthalate (PET) fibers are widely used in the preparation of artificial ligaments. However, due to their lack of hydrophilicity, PET fibers have low biocompatibility, which usually results in the poor biological activity of the products. In the present study, in order to improve the hydrophilicity and biocompatibility of PET fibers, we modified their surface using dimethylolpropionic acid (DMPA). Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), X-ray diffraction (XRD), tensile testing and cell culture were employed to observe the effects of DMPA modification on the characteristics of the DMPA-PET fibers. In contrast to the original PET fibers, the surface of the DMPA-PET fibers became rough as demonstrated by SEM. The FTIR spectrum further confirmed that a number of hydrophilic groups were formed on the surface of DMPA-PET. However, there were no significant changes in crystallinity and tensile strength between the PET and the DMPA-PET fibers as revealed by DSC and XRD (P>0.05). Finally, the cell co-culture test revealed that the adhesion and proliferation of bone marrow­derived stromal cells increased greatly on the DMPA-PET fibers compared to those on the original PET fibers (P<0.05). These results demonstrate that DMPA-PET fibers have significant potential as a material for the development of artificial ligaments.

Increased levels of calcitonin gene-related peptide in serum accelerate fracture healing following traumatic brain injury.

The mechanisms responsible for the phenomenon of an accelerated speed of fracture healing in patients with traumatic brain injury (TBI) remain unclear. The present study was performed to test the hypothesis that TBI causes changes in calcitonin gene-related peptide (CGRP) levels in sera that enhance fracture healing. A standard closed femoral fracture was produced in rats, which were subjected to additional closed head trauma. The fracture healing was assessed 4 and 8 weeks later using micro-CT. Sera, brain tissues and muscles surrounding the fracture sites collected at 24, 48, 72 and 168 h after injury were used to detect the expression of CGRP using ELISA, immunohistochemistry and RT-PCR. Micro-CT demonstrated that fracture healing and mineralization in the TBI-fracture group occurred earlier compared to the fracture-only group. ELISA analysis revealed a high concentration of CGRP in the TBI-fracture group (P<0.05), and immunohistochemistry assay and RT-PCR analysis revealed a significant increase in CGRP in the brain and muscle of the TBI-fracture group at 168 h after fracture (P<0.001). Our results indicate that the mechanism for the enhancement of fracture-healing secondary to traumatic brain injury is correlated to the high levels of CGRP, which may be released from the brain tissue into the serum.

Arthroscopic medial retinacular imbrication for the treatment of recurrent patellar instability: a simple and all-inside technique.

Proximal soft tissue realignment is the main surgical intervention for recurrent patellar instability. In recent years, all-inside arthroscopic procedures or mini-open surgeries have replaced traditional surgeries, which have more associated morbidity and poor cosmetic results. This article describes a simple and all-inside arthroscopic technique for the operative treatment of recurrent patellar instability. Using 2 epidural needles in several steps and no accessory portals required, the medial patellar retinaculum is imbricated to the desired tension. The combination of lateral release and medial retinacular placation obviously improves the patellar tracking compared with preoperatively.

Effects of different cross-linking conditions on the properties of genipin-cross-linked chitosan/collagen scaffolds for cartilage tissue engineering.

A cross-linking reagent is required to improve mechanical strength and degradation properties of biopolymers for tissue engineering. To find the optimal preparative method, we prepared diverse genipin-cross-linked chitosan/collagen scaffolds using different genipin concentrations and various cross-linking temperatures and cross-linking times. The compressive strength increased with the increasing of genipin concentration from 0.1 to 1.0%, but when concentration exceeded 1.0%, the compressive strength decreased. Similarly, the compressive strength increased with the increasing of temperature from 4 to 20°C, but when temperature reached 37°C, the compressive strength decreased. Showing a different trend from the above two factors, the effect of cross-linking time on the compressive strength had a single increasing tendency. The other results also demonstrated that the pore size, degradation rate and swelling ratio changed significantly with different cross-linking conditions. Based on our study, 1.0% genipin concentration, 20°C cross-linking temperature and longer cross-linking time are recommended.

Cartilage regeneration using adipose-derived stem cells and the controlled-released hybrid microspheres.

OBJECTIVE: This study was to evaluate the effect of hybrid microspheres (MS) composed of gelatin transforming growth factor-beta (TGF-beta1)-loaded MS and chitosan MS on the enhancement of differentiation of adipose-derived stem cells (ASCs) into chondrocytes in pellet culture in vitro and the reparative capacity of pellet from ASCs and the hybrid MS-TGF used to repair cartilage defects in vivo. METHODS: The morphology of the controlled-released MS was observed with scanning electron microscopy (SEM) and mechanical property was also tested in this study. In vitro TGF-beta1 release was evaluated by an enzyme-linked immunosorbent assay. The protein expression of Collagen II was tested by Western blot. In addition, a preliminary study on cartilage regeneration was also performed in vivo. RESULTS: When chondrogenic differentiation of ASCs in both MS was evaluated, the protein expression of Collagen II became significantly increased for the hybrid MS-TGF, as compared with the gelatin MS-TGF. Mechanical result showed that the hybrid MS was superior to the gelatin MS. Observation of histology in vivo demonstrated that the pellet from ASCs and the hybrid MS-TGF promoted cartilage regeneration in the defects of articular cartilage much better than other groups. CONCLUSION: Our study demonstrated that the pellet from ASCs and the hybrid MS-TGF can provide an easy and effective way to construct the tissue engineered cartilage in vitro and in vivo.

Enhanced chondrogenesis of adipose-derived stem cells by the controlled release of transforming growth factor-beta1 from hybrid microspheres.

BACKGROUND: Articular cartilage has a limited self-regenerative capacity, and tissue engineering is a promising solution to the problem of cartilage damage. OBJECTIVE: The aim of this study was to evaluate the effect of hybrid microspheres (MS) composed of transforming growth factor (TGF)-beta1-loaded gelatin MS and chitosan MS on enhancement of the differentiation of adipose-derived stem cells (ASCs) into chondrocytes in pellet culture. METHODS: In vitro TGF-beta1 release was evaluated by an enzyme-linked immunosorbent assay. The content of DNA and glycosaminoglycans (GAGs) was tested by biochemical methods. In addition, quantitative PCR was used to analyze the expression of collagen II and aggrecan. RESULTS: Increased proliferation of ASCs was observed in the hybrid TGF-beta1-loaded MS in comparison to the TGF-beta1-loaded gelatin MS. The chondrogenic differentiation of ASCs in both constructs was evaluated, and GAG content and the gene expression of collagen II and aggrecan were significantly higher in the hybrid TGF-beta1-loaded MS than in the TGF-beta1-loaded gelatin MS. CONCLUSIONS: Enhanced differentiation of ASCs by hybrid TGF-beta1-loaded MS may provide an easy and effective way to construct tissue-engineered cartilage.

[Anterolateral minimally-invasive total hip arthroplasty: a clinical comparative study of 110 cases].

OBJECTIVE: To explore the indications and key points of anterolateral minimally-invasive total hip arthroplasty. METHODS: 110 baseline indexes matched patients admitted for unilateral total hip arthroplasty were randomly assigned to 2 equal groups to undergo surgery through a short anterolateral incision of < or = 10 cm or a standard posterolateral incision. All operations were done by the same surgeon. The demographic data, perioperative indexes, and postoperative function indexes were recorded and statistically analyzed. RESULTS: No significant differences were detected with respect to operation time, abduction angle, anteversion angle, stem alignment, and stem fixation between these 2 groups. The incision length, blood loss, perioperative transfusion, and 100 - mm visual analogue pain scale (VAS) score at the first 24 hours of the anterolateral approach group were (7.49 +/- 0.86) cm, (376.18 +/- 168.30) ml, (410.09 +/- 136.46) ml, and (30.76 +/- 21.77) respectively, all significantly shorter, less, or lower than those of the standard posterolateral approach group [(15.2 +/- 1.8) cm, (605.0 +/- 225.1) ml, (629.5 +/- 232.9) ml, and (50.3 +/- 13.7) respectively, all P < 0.01]. The Harris hip score and Barthel index 3 months after operation of the anterolateral approach group were (83.80 +/- 5.64) and (93.45 +/- 6.37) respectively, both significantly higher than those of the standard posterolateral approach group [(75.0 +/- 7.5) and (94.6 +/- 7.5) respectively, both P < 0.01)], however, there were not significant differences in the Harris hip score and Barthel index 3 years after operation between these 2 groups. CONCLUSIONS: Fewer traumas, less blood loss and rapid recovery can be obtained through this new total hip arthroplasty approach. But experienced doctors and special instruments are prerequisite.

Fingertip replantation: determinants of survival.

BACKGROUND: The purpose of this study was to determine the risk factors for an unsuccessful replanted fingertip. METHODS: Two hundred eleven complete fingertip amputations in 211 patients who underwent replantation surgery between August of 1990 and March of 2006 were included in this study. The patients' age, gender, smoking history, digit position, dominant hand, amputation level, injury mechanism, platelet count, ischemia time, preservation method of the amputated part, anesthesia, number of arteries repaired, venous drainage, use of vein grafting, neurorrhaphy, bone shortening, and smoking after operation were tested for their impact on fingertip survival. RESULTS: One hundred seventy-two of 211 patients (81.5 percent) had a successful replantation. Univariate analysis showed crush or avulsion injury, high platelet count, and inappropriate preservation of the amputated part in saline solution or ethanol to be associated with a high incidence of replantation failure. Twenty-two of 54 patients (41 percent) who had a crush or avulsion trauma had failed replantation. Logistic regression analysis identified injury mechanism, platelet count, smoking after operation, preservation method of the amputated part, and the use of vein grafting as statistically significant predictive factors for success or failure. CONCLUSIONS: Injury mechanism, platelet count, smoking after operation, preservation method of amputated part, and the use of vein grafting were found to be the main predictors for the survival of the replanted fingertip. Applying external bleeding in zone 1 and venous drainage through the medullary cavity in zone 2 or venous anastomosis combined with vein grafting rather than venous anastomosis alone were strongly recommended in the fingertip replantation of crush or avulsion injury.

Effect of ovariectomy on BMD, micro-architecture and biomechanics of cortical and cancellous bones in a sheep model.

Osteoporotic/osteopenia fractures occur most frequently in trabeculae-rich skeletal sites. The purpose of this study was to use a high-resolution micro-computed tomography (micro-CT) and dual energy X-ray absorptionmeter (DEXA) to investigate the changes in micro-architecture and bone mineral density (BMD) in a sheep model resulted from ovariectomy (OVX). Biomechanical tests were performed to evaluate the strength of the trabecular bone. Twenty adult sheeps were randomly divided into three groups: sham group (n=8), group 1 (n=4) and group 2 (n=8). In groups 1 and 2, all sheep were ovariectomized (OVX); in the sham group, the ovaries were located and the oviducts were ligated. In all animals, BMD for lumbar spine was obtained during the surgical procedure. BMD at the spine, femoral neck and femoral condyle was determined 6 months (group 1) and 12 months (group 2) post-OVX. Lumbar spines and femora were obtained and underwent BMD scan, micro-CT analysis. Compressive mechanical properties were determined from biopsies of vertebral bodies and femoral condyles. BMD, micro-architectural parameters and mechanical properties of cancellous bone did not decrease significantly at 6 months post-OVX. Twelve months after OVX, BMD, micro-architectural parameters and mechanical properties decreased significantly. The results of linear regression analyses showed that trabecular thickness (Tb.Th) (r=0.945, R2=0.886) and bone volume fraction (BV/TV) (r=0.783, R2=0.586) had strong (R2>0.5) correlation to compression stress. In OVX sheep, changes in the structural parameters of trabecular bone are comparable to the human situation during osteoporosis was induced. The sheep model presented seems to meet the criteria for an osteopenia model for fracture treatment with respect to morphometric and mechanical properties. But the duration of OVX must be longer than 12 months to ensure the animal model can be established successfully.

Treatment of ipsilateral femoral neck and shaft fractures.

OBJECTIVE: To investigate the clinical characteristics, treatment options and causes of misdiagnosis of ipsilateral femoral neck and shaft fractures. METHODS: Among 20 patients with ipsilateral femoral neck and shaft fractures, 19 were treated operatively and 1 was treated conservatively. Sixteen cases of femoral shaft fractures were treated by open reduction and internal fixation with compressive plate, and 2 cases were treated with interlocking intramedullary nailing. Eighteen femoral neck fractures were treated with cannulated screws. Another patient was treated with proximal femoral nail to fix both the neck and shaft. Delayed diagnosis for femoral neck fractures occurred in 2 cases preoperatively. RESULTS: A total of 19 patients were followed up. The follow up period ranged from 5 to 48 months with an average of 15 months. All the fractures were healed. CONCLUSION: For case of femoral shaft fracture caused by high energy injury, an AP pelvic film should be routinely taken. Once the femoral neck fracture is recognized, operative reduction and fixation should be performed in time. Femoral neck and shaft fractures should be fixed separately.