Anisotropic Microspheres-Cryogel Composites Loaded with Magnesium l-Threonate Promote Osteogenesis, Angiogenesis, and Neurogenesis for Repairing Bone Defects.

To achieve osteogenesis, angiogenesis, and neurogenesis for repairing bone defects, we constructed an anisotropic microspheres-cryogel composite loaded with magnesium l-threonate (MgT). These composites were prepared by the photo-click reaction of norbornene-modified gelatin (GB) in the presence of MgT-loaded microspheres through the bidirectional freezing method. The composites possessed an anisotropic macroporous (around 100 µm) structure and sustained release of bioactive Mg2+, which facilitate vascular ingrowth. These composites could significantly promote osteogenic differentiation of bone marrow mesenchymal stem cells, tubular formation of human umbilical vein vessel endothelial cells, and neuronal differentiation in vitro. Additionally, these composites significantly promoted early vascularization and neurogenesis as well as bone regeneration in the rat femoral condyle defects. In conclusion, owing to the anisotropic macroporous microstructure and bioactive MgT, these composites could simultaneously promote bone, blood vessel, and nerve regeneration, showing great potential for bone tissue engineering.

Mechanisms of Hierarchical Topographies Tuning Bacteria and Cell Biological Responses to the Surfaces of Pure Titanium and Cu-Bearing Titanium Alloy.

The competition between cells integration and bacterial colonization determines the fate of implantations. To reveal the effects of clinical implant topographies on osteoblast differentiation and bacterial biofilm formation, a series of micron/submicron/nano-hierarchical structures were created at pure titanium surfaces (Ti-I, Ti-II, Ti-III). It was found that the hierarchical structures promoted MC3T3-E1 cell differentiation through contact guidance and Ti-II processed the best osteogenic ability. Undesirably, hierarchical surfaces further accelerated the biofilm formation due to submicron structures with low interaction. To reduce the risk of bacterial infections, hierarchical structures were prepared on the antibacterial Cu-bearing titanium alloy surfaces (TiCu-I, TiCu-II, TiCu-III). Hierarchical topographies not only endowed TiCu surfaces with antibacterial trapping characteristics due to CuO doped in the outermost oxides layer but also shifted the corrosion behavior of TiCu alloy into activation-passivation, increasing the Cu-ion release rate and further promoting the osteogenic differentiation. TiCu-III possessed excellent antibacterial trapping ability and optimal osteogenic action. Finally, in the osteomyelitis-modeled mice, hierarchical topographies aggravated the bacterial infection around Ti implants, which entirely lost the osseointegration, while all of the TiCu surfaces significantly inhibited the infection and accelerated the formation of new bone tunnels around the implants. In vivo studies successfully confirmed the tuning mechanism of hierarchical topographies on the biological responses of bacteria and cells to the Ti and TiCu alloys, which would pave the way to develop novel biofunctionalized metal implants.

Enhanced bone formation in rat critical-size tibia defect by a novel quercetin-containing alpha-calcium sulphate hemihydrate/nano-hydroxyapatite composite.

We developed an innovative method to include quercetin into alpha-calcium sulphate hemihydrate/nano-hydroxyapatite (α-CSH/n-HA), to prepare a novel quercetin-containing α-CSH/n-HA composite (Q-α-CSH/n-HA). The physicochemical properties, and ability of Q-α-CSH/n-HA to promote cell proliferation, migration, and osteogenic differentiation of bone marrow stem cells (BMSCs) in vitro were examined. Further, the potential of Q-α-CSH/n-HA to promote bone defect repair was studied using a Sprague-Dawley rat model of critical tibial defects. Imaging was conducted by radiography and micro-CT, and bone defect repairs were observed by histopathological staining. Addition of quercetin clearly increased the porosity of the degraded composite, which elevated the cell proliferation rate, migration ability, osteogenesis differentiation, and mineralisation of BMSCs. Further, quercetin-containing composite increased the expression levels of OSX, RUNX2, OCN, ALP, BMP-2, OPN, BSP, SMAD2, and TGF-ß in BMSCs, while it downregulated TNF-α. X-ray and micro-CT imaging showed that the quercetin-containing composite significantly enhanced bone defect repair and new bone in formation. Haematoxylin and eosin, Goldner, and Safranin O staining also showed that quercetin significantly increased new bone generation and promoted composite degradation and absorption. Moreover, immunofluorescence assay revealed that quercetin significantly increased the number of RUNX2/OSX/OCN-positive cells. Overall, our data demonstrate that Q-α-CSH/n-HA has excellent biocompatibility, bone conductivity, and osteo-induction performance in vitro and mediates enhanced overall repair effects and bone reconstruction in vivo, indicating that it is a promising artificial bone graft to promote bone regeneration.

Physicochemical and biological properties of carboxymethyl chitosan zinc (CMCS-Zn)/α­calcium sulfate hemihydrate (α-CSH) composites.

To improve the osteoinductivity, antibacterial activity, and clinical application of calcium sulfate hemihydrate (CSH), carboxymethyl chitosan zinc (CMCS-Zn) and α-CSH were prepared using different mass ratios. The setting time and injectability of the CMCS-Zn/α-CSH composite were increased with increasing CMCS-Zn content. After adding different amounts of CMCS-Zn to α-CSH, the fine lamellar structure of CMCS-Zn was found by scanning electron microscopy (SEM), which is evenly distributed in the matrix of α-CSH. With the increase of CMCS-Zn, the pores on the surface gradually increased. After mixing CMCS-Zn and α-CSH, no new phase was measured by X-ray diffraction (XRD) and Fourier transform (FTIR) spectroscopy. The degradation rate of CMCS-Zn/α-CSH decreased with increasing CMCS-Zn content, and the pH was stable during the degradation process. The release of Zn2+ increased with increasing CMCS-Zn content, while the release of Ca2+ decreased. Extracts of CMCS-Zn/α-CSH composites up-regulated the osteoinduction and migration of rat bone marrow stem cells. The antibacterial ability of CMCS-Zn/α-CSH was evaluated as a function of CMCS-Zn content. In the rat bone defect model, 5% CMCS-Zn/α-CSH group revealed a higher volume and density of trabeculae by micro-CT 8 weeks after the operation. Therefore, CMCS-Zn/α-CSH was demonstrated to be an adjustable, degradable, substitute biomaterial (with osteogenesis-promoting effects) for use in bone defects, which also has antibacterial activity that can suppress bone infection.

mRNA Transcriptome Analysis of Bone in a Mouse Model of Implant-Associated Staphylococcus aureus Osteomyelitis.

To investigate the molecular pathogenesis of bone with osteomyelitis, we developed implant-associated osteomyelitis (IAOM) models in mice. An orthopedic stainless pin was surgically placed in the right femoral midshaft of mice, followed by an inoculation of Staphylococcus aureus into the medullary cavity. Typical characteristics of IAOM, like periosteal reaction and intraosseous abscess, occurred by day 14 postinfection. By day 28 postinfection, necrotic abscess, sequestrum formation, and deformity of the whole femur were observed. Transcriptional analysis identified 101 and 1,702 differentially expressed genes (DEGs) between groups by days 3 and 14 postinfection, respectively. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed the enrichment of pathways in response to the bacterium, receptor-ligand activity, and chemokine signaling by day 3 postinfection. However, by day 14 postinfection, the enrichment switched to angiogenesis, positive regulation of cell motility and migration, skeletal system development, and cytokine-cytokine receptor interaction. Furthermore, protein-protein interaction network analysis identified 4 cytokines (interleukin 6 [IL-6], Cxcl10, gamma interferon [IFN-γ], and Cxcl9) associated with IAOM at an early stage of infection. Overall, as the pathological changes in this mouse model were consistent with those in human IAOM, our model may be used to investigate the mechanism and treatment of IAOM. Furthermore, the data for transcriptome sequencing and bioinformatic analysis will be an important resource for dissecting the molecular pathogenesis of bone with IAOM.

Copper-Containing Alloy as Immunoregulatory Material in Bone Regeneration via Mitochondrial Oxidative Stress.

In the mammalian skeletal system, osteogenesis and angiogenesis are closely linked by type H vessels during bone regeneration and repair. Our previous studies confirmed the promotion of these processes by copper-containing metal (CCM) in vitro and in vivo. However, whether and how the coupling of angiogenesis and osteogenesis participates in the promotion of bone regeneration by CCM in vivo is unknown. In this study, M2a macrophages but not M2c macrophages were shown to be immunoregulated by CCM. A CCM, 316L-5Cu, was applied to drilling hole injuries of the tibia of C57/6 mice for comparison. We observed advanced formation of cortical bone and type H vessels beneath the new bone in the 316L-5Cu group 14 and 21 days postinjury. Moreover, the recruitment of CD206-positive M2a macrophages, which are regarded as the primary source of platelet-derived growth factor type BB (PDGF-BB), was significantly promoted at the injury site at days 14 and 21. Under the stimulation of CCM, mitochondria-derived reactive oxygen species were also found to be upregulated in CD206hi M2a macrophages in vitro, and this upregulation was correlated with the expression of PDGF-BB. In conclusion, our results indicate that CCM promotes the evolution of callus through the generation of type H vessels during the process of bone repair by upregulating the expression of PDGF-BB derived from M2a macrophages.

Strain distribution of the anterolateral ligament during internal rotation at different knee flexion angles: A biomechanical study on human cadavers.

BACKGROUND: Injuries of the anterolateral ligament (ALL) are fairly common in patients with ruptures of the anterior cruciate ligament (ACL). Before considering repair or reconstruction of the ALL, the lack of knowledge with regard to the biomechanical behavior of this ligament must be considered. The purpose of this study was to analyze the strain of the ALL induced by tibial internal rotation at different flexion angles and find out the strain distribution features. METHODS: The ALLs of ten fresh-frozen cadaver knees were dissected. All specimens underwent tibial internal rotation from 0° to 25° at 30°, 60°, 90°, and 120° of knee flexion. Strain distribution of the ALL during internal rotation was recorded by digital image correlation (DIC). The overall strain and sub-regional strain were measured. RESULTS: The strain of the ALL increased with increasing tibial internal rotation. With 25° of internal rotation, the overall strain at each flexion angle was 12.89 ±â€¯2.73% (30°), 15.32 ±â€¯2.50% (60°), 18.94 ±â€¯2.34% (90°), and 20.10 ±â€¯3.27% (120°). The sub-regional strain was significantly different at all flexion angles. The strain of the distal 1/3 of the ALL was the greatest, followed by the middle 1/3, while the proximal 1/3 was the smallest (all P < 0.001). CONCLUSION: The ALL resisted internal rotation of the tibia by becoming more tense with increasing rotation. A significantly high strain was observed in the distal portion near the tibial insertion site of the ALL, which may suggest that this region is prone to injury with excessive internal rotation.

Strain Distribution in the Anterior Inferior Tibiofibular Ligament, Posterior Inferior Tibiofibular Ligament, and Interosseous Membrane Using Digital Image Correlation.

BACKGROUND: Ligament repair and augmentation techniques can stabilize syndesmosis injuries. However, little is known about the mechanical behavior of syndesmotic ligaments. The aim of this study was to analyze full-field strain, strain trend under foot rotation, and subregional strain differences of the anterior inferior tibiofibular ligament (AITFL), posterior inferior tibiofibular ligament (PITFL), and interosseous membrane (IOM). METHODS: Eleven fresh-frozen lower limbs were dissected to expose the AITFL, PITFL, and IOM. The foot underwent rotation from 0° to 25° internal and 35° external, with 3 ankle positions (neutral, 15° dorsiflexion, and 25° plantarflexion) and a vertical load of 430 N. Ligament strain was recorded using digital image correlation. RESULTS: The mean strain on the AITFL with 35° external rotation was greater in the proximal portion compared with distal portion in the neutral position ( P = .009) and dorsiflexion ( P = .003). The mean strain in the tibial insertion and midsubstance near tibial insertion were greater when compared with other regions ( P = .018 and P = .009). The subregions of mean strain in the PITFL and IOM groups were not significantly different. The strain trend of AITFL, PITFL, and IOM showed common transformation, just when the foot was externally rotated. CONCLUSION: The findings of this study show that a significantly high strain was observed on the proximal part and the midsubstance near the Chaput tubercle of the AITFL when the ankle was externally rotated. All 3 ligaments resisted the torque in the syndesmosis by external rotation of the foot. CLINICAL RELEVANCE: This study allows for better understanding of the mechanical behavior of the syndesmosis ligaments, which could influence the repair technique and AITFL augmentation techniques.