Microstructurally and mechanically tunable acellular hydrogel scaffold using carboxymethyl cellulose for potential osteochondral tissue engineering.

In tissue engineering, scaffold microstructures and mechanical cues play a significant role in regulating stem cell differentiation, proliferation, and infiltration, offering a promising strategy for osteochondral tissue repair. In this present study, we aimed to develop a facile method to fabricate an acellular hydrogel scaffold (AHS) with tunable mechanical stiffness and microstructures using carboxymethyl cellulose (CMC). The impacts of the degree of crosslinking, crosslinker length, and matrix density on the AHS were investigated using different characterization methods, and the in vitro biocompatible of AHS was also examined. Our CMC-based AHS showed tunable mechanical stiffness ranging from 50 kPa to 300 kPa and adjustable microporous size between 50 µm and 200 µm. In addition, the AHS was also proven biocompatible and did not negatively affect rabbit bone marrow stem cells' dual-linage differentiation into osteoblasts and chondrocytes. In conclusion, our approach may present a promising method in osteochondral tissue engineering.

Dynamics analysis of the anterior cruciate ligament reconstruction surgery based on magnetic resonance imaging.

In clinical practice, anterior cruciate ligament (ACL) rupture is always repaired by the single-beam reconstruction method. Before the surgery, the surgeon made the diagnosis based on medical images, such as CT (computerized tomography) and MR (magnetic resonance) images. However, little is known about how biomechanics governs the biological nature for femoral tunnel position. In the present study, three volunteers' motion trails were captured by six cameras when they were doing squat movement. The medical image can reconstruct the structure of the ligaments and bones and a left knee model was reconstructed by MIMICS by MRI data of DICOM format. Finally, the effects of different femoral tunnel positions on ACL biomechanics were characterized by the inverse dynamic analysis method. The results showed that there were significant differences in the direct mechanical effects of the anterior cruciate ligament at different locations of the femoral tunnel (p < 0.05), the peak stress of ACL in the low tension area was 1097.24 ± 25.55 N, and the peak stress of ACL in the distal femur was 356.81 ± 15.39 N, both of which were much higher than that in the direct fiber area (118.78 ± 20.68 N).

A facile inverse dynamics method to study the impacts of fatigue on the lower limb biomechanics.

Fatigue can significantly affect the biomechanics of the anterior cruciate ligament (ACL) during single-legged jumping, and these changes are closely related to ACL injuries. Unfortunately, there is no convenient way to accurately and quickly track these changes. This present study aimed to develop such a method based on video capture and inverse dynamics simulation.Are our method's results accurate and reliable? Fifteen participants performed sing-legged jumping before and after the fatigue protocol, and their actions were videotaped. The videos were processed and converted to marked motion data, used to drive the mannequin model in AnyBody Modelling System (AMS) for the inverse dynamics simulations. The ACL segment was also constructed based on one participant's MRI data and added the mannequin model.Our results were similar to the findings from previous studies. Neuromuscular fatigue decreased the peak flexion angles and increased the low-the-limb muscle strength and activation. These alterations might contribute to ACL tears and ruptures. In addition, the simulation showed that the ACL force significantly (p < 0.05) increased as a result of fatigue during single-legged jumping. Our study provides a facile and reliable method to study the effects of neuromuscular fatigue on lower-the-limb biomechanics. Such a method can be applied to investigate other risk factors on ACL injuries and assist in developing workable plans for athlete training in the future.

A biomechanical analysis of skiing-related anterior cruciate ligament injuries based on biomedical imaging technology.

Alpine skiing is an attractive but highly risky sport, and the anterior cruciate ligament (ACL) tear is one of the most common diagnoses of skiing-related injuries. To better prevent such injuries among athletes and recreational skiers, we developed a facile and reliable biomechanical method to analyze the differences between "right" and "wrong" movements during skiing and their impacts on ACL stress loading. Unlike those conventional methods that are very difficult to implement and time-consuming, our method was developed based on inverse dynamics analyses and video capture, which were much easier to implement in the real-world setting. It is shown that, with a harmful skiing action, the knee joint's maximum reaction force significantly increases compared to nonharmful skiing actions. The peak front-and-rear force increased from 1242 N to 3105 N, and the peak axial force increased from 1023 N to 3443 N, which significantly exceeded the maximum tensile loading (2000 N) in the ACL. Our results are proven to be reliable and consistent with findings obtained with other methods. This method may substitute current complex analytical methods and be easier to apply in sports-related injury-prevention applications.

The Anti-inflammatory Effect of Chitosan Oligosaccharide on Heart Failure in Mice.

Heart failure is currently one of the leading causes of death worldwide, and the inflammatory factors play an important role in its development. Chitosan oligosaccharide (COS), a low-molecular-weight form of chitosan, has many specific biological activities. In this study, COS effects on heart failure were studied for the first time by performing transverse arch constriction (TAC) surgery in mice, as an animal model of heart failure. Our findings revealed that COS administration (in both 40 mg/kg and 80 mg/kg doses) significantly ameliorated TCA-induced left ventricular (LV) hypertrophy as well as the increase in lung and heart weight in mice, while improving TAC-induced LV dysfunction. Both doses effectively attenuated LV cardiomyocyte hypertrophy, while decreasing heart inflammation after heart failure in mice. In conclusion, our results indicated that the supplementation of COS in normal diet might be an effective way to prevent further myocardial tissue damage in patients suffering from heart failure.

PDMS-enhanced slowly degradable Ca-P-Si scaffold: Material characterization, fabrication and in vitro biocompatibility study.

A slowly degradable bone scaffold can well maintain the balance between new bone regeneration and scaffold resorption, esp. for seniors or patients suffering from pathological diseases, because too fast degradation can lead to the loss of long-term biological stability and result in scaffold failure. In this present study, calcium phosphate silicate (CPS) and polydimethylsiloxane (PDMS) were blended in different ratios to formulate slurries for scaffold fabrication. The effects of crosslinked PDMS on the CPS material properties were first characterized and the most viable formulation of CPS-PDMS slurry was determined based on the aforementioned results to 3D fabricate scaffolds. The biocompatibility of CPS-PDMS was further evaluated based on the scaffold extract's cytotoxicity to osteoblast cells. Furthermore, real-time PCR was used to investigate the effects of scaffold extract to increase osteoblast proliferation. It is showed that the crosslinked PDMS interfered with CPS hydration and reduced both setting rate and compressive strength of CPS. In addition, CPS porosity was also found to increase with PDMS due to uneven water distribution as a result of increased hydrophobicity. Degradation and mineralization studies show that CPS-PDMS scaffold was slowly degradable and induced apatite formation. In addition, the in vitro analyses show that the CPS-PDMS scaffold did not exert any cytotoxic effect on osteoblast cells but could improve the cell proliferation via the TGFß/BMP signaling pathway. In conclusion, CPS-PDMS scaffold is proved to be slowly degradable and biocompatible. Further analyses are therefore needed to demonstrate CPS-PDMS scaffold applications in bone regeneration.

The impact of leuprolide acetate-loaded calcium phosphate silicate cement to bone regeneration under osteoporotic conditions.

Osteoporosis is detrimental to the health of skeletal structure and significantly increases the risks of bone fracture. Moreover, bone regeneration is adversely impaired by increased osteoclastic activities as a result of osteoporosis. In this study, we developed a novel formulation of injectable bone cement based on calcium phosphate silicate cement (CPSC) and leuprolide acetate (LA). Several combinations of LA-CPSC bone cement were characterized and, it is found that LA could increase the setting time and compressive strength of CPSC in a concentration-dependent manner. Moreover, thein vitroresults revealed that LA-CPSC was biocompatible and able to encourage the osteoblast proliferation via the mTOR signalling pathway. Furthermore, the LA-CPSC was implanted in the osteoporotic rats to evaluate its effectiveness to repair bone fractures under the osteoporotic conditions. The biomarker study and micro-CT analyses indicated that LA-CPSC could effectively reduce the osteoclast activities and promote the bone regeneration. In conclusion, our study demonstrated that LA-CPSC injectable bone cement should be a viable solution to repair bone fractures under the osteoporotic conditions.

Bioprinting of human nasoseptal chondrocytes-laden collagen hydrogel for cartilage tissue engineering.

Skin cancer patients often have tumorigenic lesions on their noses. Surgical resection of the lesions often results in nasal cartilage removal. Cartilage grafts taken from other anatomical sites are used for the surgical reconstruction of the nasal cartilage, but donor-site morbidity is a common problem. Autologous tissue-engineered nasal cartilage grafts can mitigate the problem, but commercially available scaffolds define the shape and sizes of the engineered grafts during tissue fabrication. Moreover, the engineered grafts suffer from the inhomogeneous distribution of the functional matrix of cartilage. Advances in 3D bioprinting technology offer the opportunity to engineer cartilages with customizable dimensions and anatomically shaped configurations without the inhomogeneous distribution of cartilage matrix. Here, we report the fidelity of Freeform Reversible Embedding of Suspended Hydrogel (FRESH) bioprinting as a strategy to generate customizable and homogenously distributed functional cartilage matrix engineered nasal cartilage. Using FRESH and in vitro chondrogenesis, we have fabricated tissue-engineered nasal cartilage from combining bovine type I collagen hydrogel and human nasoseptal chondrocytes. The engineered nasal cartilage constructs displayed molecular, biochemical and histological characteristics akin to native human nasal cartilage.

Calcium Phosphate Silicate and Calcium Silicate Cements Suppressing Osteoclasts Activity Through Cytokine Regulation.

Calcium phosphate silicate bone cement (CPSC) can stimulate osteoblast proliferation and promote osteogenesis, but how CPSC supress osteoclast activity through cytokine regulation is not clear. In the current study, we synthesized CPSC by incorporating monocalcium phosphate (MCP) into calcium silicate cement (CSC), and analyzed the effects of CSC and CPSC on osteoclast survival with MTT. And we found that both CSC and CPSC medium could decrease osteoclast cell viability, and flow cytometry further revealed that CSC and CPSC could inhibit osteoclast activity. To elucidate the underlying mechanism, related gene and protein level of cytokines that related to osteoclast activity were evaluted. The results demonstrated that osteoclast activity was inhibited in cells treated with cement. The effects were associated with a number of cytokines stimulated by cement. In conclusion, both CSC and CPSC seem to be good substitutes of bone replacement by inhibiting osteoclast activity; the exact mechanism of how they promote bone growth, however, needs further investigations.

miR-873 suppresses H9C2 cardiomyocyte proliferation by targeting GLI1.

MicroRNAs (miRNAs) are a class of endogenous, non-coding small RNAs that regulate the expression of target genes. Previous studies have suggested that miRNAs are key regulators in cardiovascular systems. This study investigated the role of miR-873 in H9C2 cardiomyocytes by targeting glioma-associated oncogene 1 (GLI1). miR-873 was significantly up-regulated in serum samples from congenital heart disease (CHD) patients compared with those from normal individuals. Furthermore, miR-873 over-expression suppressed H9C2 proliferation and induced cell cycle arrest. Bioinformatic algorithms revealed a predicted target site for miR-873 in the 3'-untranslated region (3'UTR) of GLI1, which was verified using a dual-luciferase reporter assay. qPCR and western blot analysis also showed that miR-873 negatively regulated GLI1 mRNA and protein expression in H9C2 cells. Conversely, GLI1 over-expression partially reversed the growth-inhibitory effect of miR-873. To summarize, our data suggest that miR-873 is a novel miRNA that regulates H9C2 cell proliferation via targeting GLI1, and miR-873 may serve as a new potential biomarker diagnosis in CHD in the future.

Osteogenic and anti-osteoporotic effects of risedronate-added calcium phosphate silicate cement.

Osteoporosis greatly impairs bone fracture restoration with bone cement because the accelerated resorption decreases the osseointegration between bone and implants. In this study, we designed a new drug delivery system based on the third generation bisphosphonate risedronate (RA) and the osteogenic calcium phosphate silicate cement (CPSC). The impact of RA on CPSC's material properties and microstructure was evaluated by different characterization methods (µCT, XRD, FTIR, SEM and gas sorption). In addition, in vitro biocompatibility of RA-added CPSC was evaluated (MTT assay, flow cytometry, real-time PCR). In an in vivo study of osteoporotic rabbits, osteoporosis- and bone resorption-related biomarkers were measured over time (ELISA) and local osteogenic and anti-osteoporotic effects investigated (x-ray, CT, histology, PCR arrays). RA decreased the setting rate and compressive strength of CPSC by impeding the hydration of calcium silicate. The overall porosity of CPSC was also decreased with RA. The RA-added CPSC was biocompatible and improved osteoblast proliferation and differentiation. The slow release of RA from CPSC reduced the prevalence of osteoporosis in rabbits and improved peri-implant bone formation and osseointegration. In conclusion, RA-containing CPSC demonstrates its potentials to improve fractural restoration under osteoporotic conditions and should be further engineered to increase its effectiveness in fractural restoration.

A Comprehensive Study of Osteogenic Calcium Phosphate Silicate Cement: Material Characterization and In Vitro/In Vivo Testing.

Vertebral compression fractures can be successfully restored by injectable bone cements. Here the as-yet unexplored in vitro cytotoxicity, in vivo biodegradation, and osteoconductivity of a new calcium phosphate silicate cements (CPSC) are studied, where monocalcium phosphate (MCP; 5, 10, and 15 wt%) is added to calcium silicate cement (CSC). Setting rate and compressive strength of CPSC decrease with the addition of MCP. The crystallinity, microstructure, and porosity of hardened CPSC are evaluated by X-ray diffractometer, Fourier transform infrared spectroscopy, and microcomputed tomography (CT). It is found that MCP reacts with calcium hydroxide, one of CSC hydration products, to precipitate apatite. While the reaction accelerates the hydration of CSC, the formation of calcium silicate hydrate gel is disturbed and highly porous microstructures form, resulting in weaker compressive strength. In vitro studies demonstrate that CPSC is noncytotoxic to osteoblast cells and promotes their proliferation. In the rabbit tibia implantation model, clinical X-ray and CT scans demonstrate that CPSC biodegrades slower and osseointegrates better than clinically used calcium phosphate cement (CPC). Histological studies demonstrate that CPSC is osteoconductive and induces higher bone formation than CPC, a finding that might warrant future clinical studies.

Fabrication and characterization of a novel carbon fiber-reinforced calcium phosphate silicate bone cement with potential osteo-inductivity.

The repair of bone defects is still a pressing challenge in clinics. Injectable bone cement is regarded as a promising material to solve this problem because of its special self-setting property. Unfortunately, its poor mechanical conformability, unfavorable osteo-genesis ability and insufficient osteo-inductivity seriously limit its clinical application. In this study, novel experimental calcium phosphate silicate bone cement reinforced by carbon fibers (CCPSC) was fabricated and characterized. First, a compressive strength test and cell culture study were carried out. Then, the material was implanted into the femoral epiphysis of beagle dogs to further assess its osteo-conductivity using a micro-computed tomography scan and histological analysis. In addition, we implanted CCPSC into the beagles' intramuscular pouches to perform an elementary investigation of its osteo-inductivity. The results showed that incorporation of carbon fibers significantly improved its mechanical properties. Meanwhile, CCPSC had better biocompatibility to activate cell adhesion as well as proliferation than poly-methyl methacrylate bone cement based on the cell culture study. Moreover, pronounced biodegradability and improved osteo-conductivity of CCPSC could be observed through the in vivo animal study. Finally, a small amount of osteoid was found at the heterotopic site one month after implantation which indicated potential osteo-inductivity of CCPSC. In conclusion, the novel CCPSC shows promise as a bioactive bone substitute in certain load-bearing circumstances.

Role of platelet derived growth factor (PDGF) in reverting neuronal nuclear and soma size alterations in NSC-34 cells exposed to cerebrospinal fluid from amyotrophic lateral sclerosis patients.

PURPOSE: Amyotrophic lateral sclerosis (ALS) or motor neuron disease is an adult-onset progressive neurodegenerative disorder. ALS-CSF has been shown to produce toxic effects on motor neuron cells like aberrant neurofilament phosphorylation and morphological alterations of nuclear and soma size. Our current study was designed to investigate the neuroprotective role of platelet derived growth factor (PDGF) in reverting the adverse effects produced by ALS-CSF. METHODS: Our present study was carried out to determine the restorative potential of PDGF on the toxic effects of ALS-CSF on NSC motor neuron cells from patients. Therefore the cells were divided in to three groups: (a) normal control (NC) - the cells were not exposed to ALS-CSF; (b) ALS group - the cells were exposed to ALS-CSF; (c) NALS group - the cells were exposed to non ALS CSF. Further each of these groups was supplemented with PDGF. RESULTS AND CONCLUSIONS: We observed that the mean area of nucleus and cell soma of the differentiated NSC motor neuron cells was significantly reduced in the cells exposed to ALS-CSF. We also observed that subsequent treatment with PDGF restored the soma area and nucleus of the ALS-CSF exposed cells significantly. Taken together, we show that supplementation with PDGF restores the morphological changes induced by ALS-CSF and PDGF may play a significant role in protecting motor neurons from apoptosis in ALS and thereby it promoting the cell survival.

Screening for in vitro metabolites of kakkalide and irisolidone in human and rat intestinal bacteria by ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry.

Kakkalide and irisolidone, the main isoflavones of Flos Puerariae, exhibit a wide spectrum of bioactivities. Intestinal bacteria biotransformation plays an important role in the metabolic pathways of flavones, and is directly related to the bioactivities of the prodrugs after oral administration. To the best of our knowledge, the metabolic pathways of kakkalide and irisolidone in vitro have not been comprehensively studied yet. This paper describes the strategy using ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF MS) for the rapid analysis of the metabolic profiles of kakkalide and irisolidone after incubated with human and rat intestinal bacteria. Bacteria incubated samples were prepared and analyzed after incubated under anaerobic conditions for 48 h. A total of 17 metabolites, including parent compounds, were detected in human and rat intestinal bacteria incubated samples. The results obtained indicate that hydrolysis, dehydroxylation, demethoxylation, demethylation, hydroxylation, decarbonylation, and reduction were the detected metabolic pathways of kakkalide and irisolidone in vitro. The conversion rate of irisolidone in human and rat bacteria was 8.57% and 6.51%, respectively. Biochanin A was the relatively main metabolite of irisolidone, and the content of biochanin A in human and rat bacteria was 3.68% and 4.25%, respectively. The conversion rate of kakkalide in human and rat bacteria was 99.92% and 98.58%, respectively. Irisolidone was the main metabolite of kakkalide, and the content of irisolidone in human and rat bacteria was 89.58% and 89.38%, respectively. This work not only provides the evidence of kakkalide and irisolidone metabolites in vivo, but also demonstrates a simple, fast, sensitive, and inexpensive method for identification of metabolites of other compounds transformed by intestinal bacteria.

Preparation, characterization, release kinetics, and in vitro cytotoxicity of calcium silicate cement as a risedronate delivery system.

Injectable bone cements have been well characterized and studied in non-load bearing bone fixation and bone screw augmentation applications. Current calcium phosphate cement or poly(methyl methacrylate) cement have drawbacks like low mechanical strength and in situ exothermic properties. This leads especially in patients with osteoporosis to worsening contact between implant and bone and can finally lead to implant failure. To improve these properties, a calcium silicate cement (CSC) was prepared, which additionally contained the bisphosphonate risedronate (RA) to promote osteoblast function. Cement setting rate and compressive strength were measured and found to be reduced by RA above 0.5 wt%. X-ray diffraction, Rietveld refinement analysis, scanning electron microscopy, and porosity measurements by gas sorption revealed that RA reduces calcium silicate hydrate gel formation and changes the cement's microstructure. Cumulative release profiles of RA from CSC up to 6 months into phosphate buffer solution were analyzed by high-performance liquid chromatography, and the results were compared with theoretical release curves obtained from the Higuchi equation. Fourier transform infrared spectra measurements and drug release studies indicate that calcium-RA formed within the cement, from which the drug can be slowly released over time. An investigation of the cytotoxicity of the RA-CSC systems upon osteoblast-like cells showed no toxic effects of concentrations up to 2%. The delivery of RA from within a CSC might thus be a valuable and biocompatible new approach to locally deliver RA and to reconstruct and/or repair osteoporosis-related bone fractures.

Simultaneous determination of tectorigenin and its metabolites in rat plasma by ultra performance liquid chromatography/quadrupole time-of-flight mass spectrometry.

Tectorigenin is a major isoflavone found in the flowers of Pueraria thomsonii Benth. and the rhizomes of Belamcanda chinensis (L.) DC. It possesses hepatoprotective, estrogenic, hypoglycemic and anti-inflammatory activities. In the present study, the plasma pharmacokinetic profile of tectorigenin in rats was evaluated. We developed a selective and accurate U-HPLC/Q-TOFMS method for the simultaneous characterization of nine tectorigenin metabolites, and quantitation of six major metabolites in rat plasma, including tectorigenin-7-O-glucuronide-4'-O-sulfate (Te-7G-4'S), tectorigenin-di-O-sulfate (Te-diS), tectorigenin-7-O-glucuronide (Te-7G), tectorigenin-4'-O-glucuronide (Te-4'G), tectorigenin-7-O-sulfate (Te-7S) and tectorigenin after oral administration of tectorigenin (130mg/kg). The plasma concentrations reached maximal values of 6.20±2.05µmol/L at 0.96±0.68h for Te-7G-4'S, 4.42±1.36µmol/L at 1.92±2.15h for Te-diS, 33.50±4.89µmol/L at 0.75±0.67h for Te-7G, 3.28±1.01µmol/L at 0.75±0.67h for Te-4'G, 12.80±2.80µmol/L at 0.85±1.54h for Te-7S, and 12.0±0.63µmol/L at 0.23±0.15h for tectorigenin, respectively. Enterohepatic recirculation resulted in double or triple peaks concentration curve/time profiles of the metabolites. Since the total plasma concentrations of tectorigenin conjugated metabolites were much higher than that of the tectorigenin aglycone, an extensive phase II metabolism plays an important role in the pharmacokinetics of tectorigenin in vivo.

Comparative study of fourteen alkaloids from Uncaria rhynchophylla hooks and leaves using HPLC-diode array detection-atmospheric pressure chemical ionization/MS method.

The purpose of the study is to compare alkaloid profile of Uncaria rhynchophylla hooks and leaves. Ten oxindole alkaloids and four glycosidic indole alkaloids were identified using HPLC-diode array detection (DAD) or LC-atmospheric pressure chemical ionization (APCI)-MS method, and a HPLC-UV method for simultaneous quantification of major alkaloids was validated. The hooks are characterized by high levels of four oxindole alkaloids rhynchophylline (R), isorhynchophylline (IR), corynoxeine (C) and isocorynoxeine (IC), while the leaves contained high level of two glycosidic indole alkaloids vincoside lactam (VL) and strictosidine (S). The presented methods have proven its usefulness in chemical characterization of U. rhynchophylla hooks and leaves.