Role of tissue engineered collagen based tridimensional implant on the healing response of the experimentally induced large Achilles tendon defect model in rabbits: a long term study with high clinical relevance

Background

Tendon injury is one of the orthopedic conditions poses with a significant clinical challenge to both the surgeons and patients. The major limitations to manage these injuries are poor healing response and development of peritendinous adhesions in the injured area. This study investigated the effectiveness of a novel collagen implant on tendon healing in rabbits.

Results

Seventy five mature White New-Zealand rabbits were divided into treated (n = 55) and control (n = 20) groups. The left Achilles tendon was completely transected and 2 cm excised. The defects of the treated animals were filled with collagen implants and repaired with sutures, but in control rabbits the defects were sutured similarly but the gap was left untreated. Changes in the injured and normal contralateral tendons were assessed weekly by measuring the diameter, temperature and bioelectrical characteristics of the injured area. Clinical examination was done and scored. Among the treated animals, small pilot groups were euthanized at 5, 10, 15, 20, 30, 40 and 60 (n = 5 at each time interval) and the remainder (n = 20) and the control animals at 120 days post injury (DPI). The lesions of all animals were examined at macroscopic and microscopic levels and the dry matter content, water delivery and water uptake characteristics of the lesions and normal contralateral tendons of both groups were analyzed at 120 DPI.No sign of rejection was seen in the treated lesions. The collagen implant was invaded by the inflammatory cells at the inflammatory phase, followed by fibroplasia phase in which remnant of the collagen implant were still present while no inflammatory reaction could be seen in the lesions. However, the collagen implant was completely absorbed in the remodeling phase and the newly regenerated tendinous tissue filled the gap. Compared to the controls, the treated lesions showed improved tissue alignment and less peritendinous adhesion, muscle atrophy and fibrosis. They also showed significantly better clinical scoring, indices for water uptake and water absorption, and bioelectrical characteristics than the controls.

Conclusion

This novel collagen implant was biodegradable, biocompatible and possibly could be considered as a substitute for auto and allografts in clinical practice in near future.     

Differentiation of human adipose-derived mesenchymal stem cell into insulin-producing cells: an in vitro study

Stem cells with the ability to differentiate into insulin-producing cells (IPCs) are becoming the most promising therapy for diabetes mellitus and reduce the major limitations of availability and allogeneic rejection of beta cell transplantations. Mesenchymal stem cells (MSCs) are pluripotent stromal cells with the ability to proliferate and differentiate into a variety of cell types including endocrine cells of the pancreas. This study sought to inspect the in vitro differentiation of human adipose-derived tissue stem cells into IPCs which could provide an abundant source of cells for the purpose of diabetic cell therapy in addition to avoid immunological rejection. Adipose-derived MSCs were obtained from liposuction aspirates and induced to differentiate into insulin-secreting cells under a three-stage protocol based on a combination of low-glucose DMEM medium, β-mercaptoethanol, and nicotinamide for pre-induction and high-glucose DMEM, β-mercaptoethanol, nicotinamide, and exendin-4 for induction stages of differentiation. Differentiation was evaluated by the analysis of morphology, dithizone staining, RT-PCR, and immunocytochemistry. Morphological changes including typical islet-like cell clusters were observed by phase-contrast microscope at the end of differentiation protocol. Based on dithizone staining, differentiated cells were positive and undifferentiated cells were not stained. Furthermore, RT-PCR results confirmed the expression of insulin, PDX1, Ngn3, PAX4, and GLUT2 in differentiated cells. Moreover, insulin production by the IPCs was confirmed by immunocytochemistry analysis. It is concluded that adipose-derived MSCs could differentiate into insulin-producing cells in vitro.     

Bacterial lipopolysaccharides form procollagen-endotoxin complexes that trigger cartilage inflammation and degeneration: implications for the development of rheumatoid arthritis

Introduction

We have previously reported that bacterial toxins, especially endotoxins such as lipopolysaccharides (LPS), might be important causative agents in the pathogenesis of rheumatoid arthritis (RA) in an in vitro model that simulates the potential effects of residing in damp buildings. Since numerous inflammatory processes are linked with the nuclear factor-κB (NF-κB), we investigated in detail the effects of LPS on the NF-κB pathway and the postulated formation of procollagen-endotoxin complexes.

Methods

An in vitro model of human chondrocytes was used to investigate LPS-mediated inflammatory signaling.

Results

Immunoelectron microscopy revealed that LPS physically interact with collagen type II in the extracellular matrix (ECM) and anti-collagen type II significantly reduced this interaction. BMS-345541 (a specific inhibitor of IκB kinase (IKK)) or wortmannin (a specific inhibitor of phosphatidylinositol 3-kinase (PI-3K)) inhibited the LPS-induced degradation of the ECM and apoptosis in chondrocytes. This effect was completely inhibited by combining BMS-345541 and wortmannin. Furthermore, BMS-345541 and/or wortmannin suppressed the LPS-induced upregulation of catabolic enzymes that mediate ECM degradation (matrix metalloproteinases-9, -13), cyclooxygenase-2 and apoptosis (activated caspase-3). These proteins are regulated by NF-κB, suggesting that the NF-κB and PI-3K pathways are involved in LPS-induced cartilage degradation. The induction of NF-κB correlated with activation of IκBα kinase, IκBα phosphorylation, IκBα degradation, p65 phosphorylation and p65 nuclear translocation. Further upstream, LPS induced the expression of Toll-like receptor 4 (TLR4) and bound with TLR4, indicating that LPS acts through TLR4.

Conclusion

These results suggest that molecular associations between LPS/TLR4/collagen type II in chondrocytes upregulate the NF-κB and PI-3K signaling pathways and activate proinflammatory activity.     

Synthesis of Potential Pyrimidine Derivatives via Suzuki Cross-Coupling Reaction as HIV and Kinesin Eg5 Inhibitors

A series of 4-amino-5-((4-chlorophenyl)diazenyl)-6-(alkylamino)-1-methylpyrimidin-2-one deri- vatives 7–16 were prepared by nucleophilic displacement of 6-chloro-pyrimidine 6 by various amines. 4-Amino-5-((aryl-4-yl)diazenyl)-6-aryl-1-methylpyrimidin-2-one analogs 19–27, as well as 4-amino-5-((aryl-[1,1′-biphenyl]-4-yl)diazenyl)-6-aryl-1-methylpyrimidin-2-one 29–31 and 4-amino-6-aryl-1-methylpyrimidin-2-one 34–34, were synthesized via Suzuki cross-coupling reaction, using Pd(PPh₃)₄ as a catalyst and arylboronic acids as reagents. All compounds were evaluated for their antiviral activity against the replication of HIV-1 and HIV-2 in MT-4. Compounds 6, 16, 27, and 29 showed a 50% effective concentration of >2.15, >3.03, >2.29, and >1.63 μM, respectively, but no selectivity was observed (selectivity index < 1). Two of the newly synthesized pyrimidines 12 and 29 exhibited moderate kinesin Eg5 inhibition.     

Microwave-Assisted Synthesis of Acyclic C-Nucleosides from 1,2- and 1,3-Diketones

A simple, rapid and regioselective approach for the synthesis of C-acyclic nucleosides 3, 4, 6, and 9 of dihydropyrimidine, imidazole and indeno[1,2-b]pyridine-9-one derived from 1,2- and 1,3-diketones was performed. By using DMF or pyridine as solvent or bentonite clay as a support, in the presence of TMSTf, ZnCl₂, NH₄OAc, or NH₄NO₃, all the desired products were obtained within 5–25 minutes under microwave irradiation (MWI). Acid hydrolysis of 6 and 9 afforded the free acyclic C-nucleosides 7 and 10, respectively. Upon treatment with NaOMe under MWI, 3 and 14 rearranged to the C-nucleoside 4 and 16.     

Photodegradation and Stabilization of Betamethasone-17 Valerate in Aqueous/Organic Solvents and Topical Formulations

The effects of solvent [acetonitrile, methanol, and acetonitrile/water mixture (20:80, v/v)], buffer concentration (phosphate buffer, pH 7.5), ionic strength and commonly employed adjuvants on the photodegradation of betamethasone-17 valerate in cream and gel formulations have been studied on exposure to UV light (300–400 nm). A validated high-performance liquid chromatography method has been used to determine the parent compound and its photodegraded products. The photodegradation data in the studied solvents showed greater decomposition of the drug in solvents with a lower dielectric constant. A comparatively higher rate of photodegradation was observed in the cream formulation compared to that for the gel formulation. The kinetic treatment of the photodegradation data revealed that the degradation of the drug follows first-order kinetics and the apparent first-order rate constants for the photodegradation reactions, in the media studied, range from 1.62 to 11.30 × 10⁻³ min⁻¹. The values of the rate constants decrease with increasing phosphate concentration and ionic strength which could be due to the deactivation of the excited state and radical quenching. The second-order rate constant (k′) for the phosphate ion-inhibited reactions at pH 7.5 has been found to be 5.22 × 10⁻² M⁻¹ s⁻¹. An effective photostabilization of the drug has been achieved in cream and gel formulations with titanium dioxide (33.5–42.5%), vanillin (21.6–28.7%), and butyl hydroxytoluene (18.2–21.6%).Key words: betamethasone-17 valerate, creams and gels, kinetics, photodegradation, photostabilization     

Correction for Irrelevant Absorption in Multicomponent Spectrophotometric Assay of Riboflavin, Formylmethylflavin, and Degradation Products: Kinetic Applications

In the spectrophotometric assay of multicomponent systems involved in drug degradation studies, some minor or unknown degradation products may be present. These products may interfere in the assay and thus invalidate the results due to their absorption in the range of analytical wavelengths. This interference may be eliminated by the application of an appropriate correction procedure to obtain reliable data for kinetic treatment. The present study is based on the application of linear and non-linear irrelevant absorption corrections in the multicomponent spectrophotometric assay of riboflavin and formylmethylflavin during the photolysis and hydrolysis studies. The correction procedures take into account the interference caused by minor or unknown products and have shown considerable improvement in the assay data in terms of the molar balance. The treatment of the corrected data has led to more accurate kinetic results in degradation studies.     

Theophylline Cocrystals Prepared by Spray Drying: Physicochemical Properties and Aerosolization Performance

The purpose of this work was to characterize theophylline (THF) cocrystals prepared by spray drying in terms of the physicochemical properties and inhalation performance when aerosolized from a dry powder inhaler. Cocrystals of theophylline with urea (THF-URE), saccharin (THF-SAC) and nicotinamide (THF-NIC) were prepared by spray drying. Milled THF and THF-SAC cocrystals were also used for comparison. The physical purity, particle size, particle morphology and surface energy of the materials were determined. The in vitro aerosol performance of the spray-dried cocrystals, drug-alone and a drug-carrier aerosol, was assessed. The spray-dried particles had different size distributions, morphologies and surface energies. The milled samples had higher surface energy than those prepared by spray drying. Good agreement was observed between multi-stage liquid impinger and next-generation impactor in terms of assessing spray-dried THF particles. The fine particle fractions of both formulations were similar for THF, but drug-alone formulations outperformed drug-carrier formulations for the THF cocrystals. The aerosolization performance of different THF cocrystals was within the following rank order as obtained from both drug-alone and drug-carrier formulations: THF-NIC > THF-URE > THF-SAC. It was proposed that micromeritic properties dominate over particle surface energy in terms of determining the aerosol performance of THF cocrystals. Spray drying could be a potential technique for preparing cocrystals with modified physical properties.

Electronic supplementary material

The online version of this article (doi:10.1208/s12249-012-9883-3) contains supplementary material, which is available to authorized users.Key words: aerodynamic diameter, cocrystal, spray drying, surface energy, theophylline     

Longitudinal Analysis of Maternal Plasma Apolipoproteins in Pregnancy: A Targeted Proteomics Approach

Minimally invasive diagnostic tests are needed in obstetrics to identify women at risk for complications during delivery. The apolipoproteins fluctuate in complexity and abundance in maternal plasma during pregnancy and could be incorporated into a blood test to evaluate this risk. The objective of this study was to examine the relative plasma concentrations of apolipoproteins and their biochemically modified subtypes (i.e. proteolytically processed, sialylated, cysteinylated, dimerized) over gestational time using a targeted mass spectrometry approach. Relative abundance of modified and unmodified apolipoproteins A-I, A-II, C-I, C-II, and C-III was determined by surface-enhanced laser desorption/ionization-time of flight-mass spectrometry in plasma prospectively collected from 11 gravidas with uncomplicated pregnancies at 4–5 gestational time points per patient. Apolipoproteins were readily identifiable by spectral pattern. Apo C-III₂ and Apo C-III₁ (doubly and singly sialylated Apo C-III subtypes) increased with gestational age (r²>0.8). Unmodified Apo A-II, Apo C-I, and Apo C-III₀ showed no correlation (r² = 0.01–0.1). Pro-Apo C-II did not increase significantly until third trimester (140 ± 13% of first trimester), but proteolytically cleaved, mature Apo C-II increased in late pregnancy (702 ± 130% of first trimester). Mature Apo C-II represented 6.7 ± 0.9% of total Apo C-II in early gestation and increased to 33 ± 4.5% in third trimester. A label-free, semiquantitative targeted proteomics approach was developed using LTQ-Orbitrap mass spectrometry to confirm the relative quantitative differences observed by surface-enhanced laser desorption/ionization-time of flight-mass spectrometry in Apo C-III and Apo C-II isoforms between first and third trimesters. Targeted apolipoprotein screening was applied to a cohort of term and preterm patients. Modified Apo A-II isoforms were significantly elevated in plasma from mothers who delivered prematurely relative to term controls (p = 0.02). These results support a role for targeted proteomics profiling approaches in monitoring healthy pregnancies and assessing risk of adverse obstetric outcomes.The maternal physiology during pregnancy is characterized by inflammation and hyperlipidemia. Plasma protein composition fluctuates dynamically throughout gestation to reflect these physiological changes. Apolipoproteins, a diverse subset of triglyceride transport proteins, contribute to the hyperlipidemia of pregnancy by modulating lipid homeostasis in maternal plasma (1–3). Exaggerated hyperlipidemia and peripheral apolipoprotein burden are associated with inflammatory insult and signal obstetric complications (4–5). Numerous post-translationally modified apolipoprotein isoforms are reported in plasma, but it is unclear how these modifications affect apolipoprotein function and plasma distribution. For example, changes in the glycosylation status of apolipoprotein variants predate the onset of clinical symptoms in patients with preeclampsia, a hypertensive disorder of pregnancy with clinical features in common with cardiovascular disease (6–8). The identification and functional characterization of plasma apolipoprotein isoforms and their post-translationally modified subtypes may reveal important diagnostic and/or therapeutic targets for hypertensive disorders of pregnancy (6).Mass spectrometry and targeted proteomics analyses afford unprecedented sensitivity and specificity for detecting apolipoproteins and their numerous isoforms and subtypes (9–12). Mass spectrometry approaches overcome limitations inherent in biochemical approaches (e.g. ELISA [enzyme-linked immunosorbant assays] and Western blot analysis), especially the lack of specificity of antibodies for post-translationally modified variants of plasma proteins. The objective of this study was to longitudinally evaluate maternal plasma apolipoprotein profile over gestational time by SELDI-TOF-MS (surface-enhanced laser desorption/ionization-time of flight-mass spectrometry)¹ analysis of intact proteins and a complementary targeted LTQ-Orbitrap XL MS approach. We evaluate changes in 13 post-translationally modified subtypes of the plasma apolipoproteins A-II, C-I, C-II, and C-III over gestational time.     

Lipocalin-2-mediated upregulation of various antioxidants and growth factors protects bone marrow-derived mesenchymal stem cells against unfavorable microenvironments

Despite many advantages of mesenchymal stem cells (MSCs) that make them suitable for cell therapy purposes, their therapeutic application has been limited due to their susceptibility to several stresses (e.g., nutrient-poor environment, oxidative stress, and hypoxic and masses of cytotoxic factors) to which they are exposed during their preparation and following transplantation. Hence, reinforcing MSCs against these stresses is a challenge for both basic and clinician scientists. Recently, much attention has been directed toward equipping MSCs with cytoprotective factors to strengthen them against unfavorable microenvironments. Here, we engineered MSCs with lipocalin 2 (Lcn2), a cytoprotective factor that is naturally induced following exposure of cells to stresses imposed by the microenvironment. Lcn2 overexpression not only did not interfere with the multidifferentiation capacity of the MSCs but also granted many protective properties to them. Lcn2 potentiated MSCs to withstand oxidative, hypoxia, and serum deprivation (SD) conditions via antagonizing their induced cytotoxicity and apoptosis. Adhesion rate of MSCs to coated culture plates was also enhanced by Lcn2 overexpression. In addition, Lcn2 induced antioxidants and upregulated some growth factors in MSCs. Our findings suggested a new strategy for prevention of graft cell death in MSC-based cell therapy.