Nerve Defect Treatment with a Capping Hydroxyethyl Cellulose/Soy Protein Isolate Sponge Conduit for Painful Neuroma Prevention.

Painful neuroma, as one of the complications of nerve injury from disease or trauma, results in instinctive neuropathic pain that adversely affects a patient's quality of life. To intercept neuroma development, capping strategies have been performed as effective therapies. Nonetheless, the most appropriate biocompatible material to shield the nerves is an urgent clinical requirement. Herein, a compatible hydroxyethyl cellulose (HEC)/soy protein isolate (SPI) sponge capping conduit (HSSC) is used to prevent neuroma in vivo. Following capping on the sciatic nerve stump in vivo, the behavior of the rats and the structure of tissues are compared through histological assessment and autotomy scoring. The HSSCs gained a dismal autotomy score and enhanced the amelioration, where inflammatory invasions and overdeposition of collagen are defeated. The expression of myelin growth linked genes (Krox20, MPZ, and MAG) in the HSSC group at the eighth week was almost 2 times higher than that of the no capping group. The HSSC conduit served as a physical barrier to repress the infiltration of inflammation as well as provided an optimum microenvironment for facilitating nerve rejuvenation and intercepting neuroma development during nerve amelioration.

Composite Hydrogel Conduit Incorporated with Platelet-Rich Plasma Improved the Regenerative Microenvironment for Peripheral Nerve Repair.

Peripheral nerve regeneration and functional recovery remain major challenges in clinical practice. Nerve guidance conduits (NGCs) which can regulate the regenerative microenvironment are beneficial for peripheral nerve repair. Platelet-rich plasma (PRP) can secrete multiple growth factors to regulate the regenerative microenvironment. However, current administration methods of PRP are rapidly activated followed by the burst release of growth factors, causing low therapeutic efficiency in vivo. To overcome these disadvantages, a composite nerve conduit was fabricated by incorporating PRP into a gelatin methacrylate (GelMA) and sodium alginate (SA) hydrogel. The GelMA/SA-3/PRP-20 NGCs possess optimal mechanical properties, degradation rate, and superior biological performance. Importantly, GelMA/SA-3/PRP-20 NGCs achieved the sustained release of two major growth factors (VEGF-A, PDGF-BB) from PRP. Moreover, the GelMA/SA-3/PRP-20 NGCs significantly promoted the migration of Schwann cells and the neovascularization of endothelial cells in vitro. While bridging 10 mm rat sciatic nerve defects, the GelMA/SA-3/PRP-20 NGCs promoted axonal regeneration and functional recovery of peripheral nerves. Therefore, the GelMA/SA-3/PRP-20 NGCs could regulate the regenerative microenvironment by sustained release of growth factors from PRP and shed new light on the clinical application of PRP in peripheral nerve repair.

Antibacterial Soy Protein Isolate Prepared by Quaternization.

Soy protein isolate (SPI) is green, high-yield natural plant protein, which is widely applied in industry (packing material and adhesives) and tissue engineering. It is meaningful to improve the antibacterial property of soy protein isolate to fabricate versatile safe products to meet people's requirements. In this study, quaternized soy protein isolate (QSPI) was synthesized by the reaction between 2,3-epoxypropyltrimethylammonium chloride (EPTMAC) and SPI. The positive charged (17.8 ± 0.23 mV) quaternary ammonium groups endow the QSPI with superior antibacterial properties against multiple bacteria in vitro and in vivo. Notably, QSPI maintains its good biocompatibility and promotes bacterial-infected wound healing in rat models. Furthermore, QSPI possesses superior water solubility in a broad pH range than raw SPI. Altogether, this soy protein isolate derivative with antibacterial property and superior water solubility may extend the application of SPI in industry and tissue engineering.

Secretome of Mesenchymal Stem Cells from Consecutive Hypoxic Cultures Promotes Resolution of Lung Inflammation by Reprogramming Anti-Inflammatory Macrophages.

The secretome from hypoxia-preconditioned mesenchymal stem cells (MSCs) has been shown to promote resolution of inflammation and alleviate acute lung injury (ALI) through its immunomodulatory function. However, the effects of consecutive hypoxic culture on immunomodulatory function of the MSCs secretome are largely unclarified. Here, we intend to investigate the effects of consecutive hypoxia on therapeutic efficacy of conditioned medium derived from MSCs (MSCs-CM) in alleviating ALI. Human umbilical cord-derived MSCs (UC-MSCs) were consecutively cultured in 21% O2 (Nor-MSCs) or in 1% O2 (Hypo-MSCs) from passage 0. Their conditioned medium (Nor-CM and Hypo-CM respectively) was collected and administered into ALI models. Our findings confirmed that Hypo-MSCs exhibited increased proliferation ability and decreased cell senescence compared with Nor-MSCs. Consecutive hypoxia promoted UC-MSCs to secrete immunomodulatory cytokines, such as insulin-like growth factor 1(IGF1), IL10, TNFα-stimulated gene 6(TSG6), TGFß, and prostaglandin E2 (PGE2). Both Nor-CM and Hypo-CM could effectively limit lung inflammation, promote efferocytosis and modulate anti-inflammatory polarization of lung macrophages in ALI models. Moreover, the effects of Hypo-CM were more potent than Nor-CM. Taken together, our findings indicate that consecutive hypoxic cultures could not only promote both proliferation and quality of UC-MSCs, but also enhance the therapeutic efficacy of their secretome in mitigating lung inflammation by promoting efferocytosis and anti-inflammatory polarization of macrophages.

Facile fabrication of soy protein isolate-functionalized nanofibers with enhanced biocompatibility and hemostatic effect on full-thickness skin injury.

Extensive full-thickness skin defect lacks self-healing ability. Tissue engineering wound dressing is considered as the most promising approach to promote wound healing. In this study, a series of biocompatible and hemostatic nanofiber dressings were fabricated. Soy protein isolate (SPI) and poly(L-lactic acid) (PLLA) solutions were mixed in certain proportions for high-voltage electrospinning. The obtained products were coded as SPNF-n (n = 100, 80, 60 and 40, corresponding to the weight percentage of PLLA solution). We found that SPNF-n (n = 100, 80, 60 and 40) could facilitate the adhesion and spread of L929 cells. In particular, SPNF-80 was capable of promoting fibroblast proliferation and diminishing inflammation. Compared with the neat PLLA film (SPNF-100), the biosafety and hemostatic effect of SPNF-80 got significantly improved. The hemostatic effect of SPNF-80 was comparable with that of a commercial gelatin sponge. In vivo wound healing assay demonstrated that SPNF-80 could accelerate the wound healing process by enhancing vascularization, re-epithelization and collagen formation. In conclusion, our results reveal that SPNF-n has good biocompatibility and hemostatic effect, and exhibits great application potential in wound healing.

Natural Flammulina velutipes-Based Nerve Guidance Conduit as a Potential Biomaterial for Peripheral Nerve Regeneration: In Vitro and In Vivo Studies.

The treatment and repair of serious peripheral nerve injuries remain challenging in the clinical practice, while the application of multifunctional nerve guidance conduits (NGCs) based on naturally derived polymers has attracted much attention in recent years because of their excellent physicochemical properties and biological characteristics. Flammulina velutipes (Curt. ex FV) is a popular edible mushroom characterized by hollow tubular structures, antibacterial activities, and high nutritional properties. In this study, FV is utilized to construct NGCs (labeled FVC) via a freeze-drying technique without chemical modifications. The morphology, physical properties, cellular biocompatibility, antibacterial properties, and nerve regeneration capacity of FVC were assessed both in vitro and in vivo. FVC is composed of hollow tubes and evenly irregular interconnected micropores with 73.8 ± 5.5% porosity and 476.1 ± 12.9 µm hollow tube diameter. The inner surface of the FVC presents multiple microgrooves elongated parallel to the long axis. Moreover, FVC possessed strong antibacterial activity and could inhibit Gram-positive Staphylococcus aureus growth by up to 96.0% and Gram-negative Escherichia coli growth by up to 94.8% in vitro. FVC exhibited excellent biocompatibility and effectively promoted PC-12 cell proliferation and elongation in vitro. When applied to repair critical-sized sciatic nerve defects, FVC could effectively stimulate nerve functional recovery and axonal outgrowth in a rat model. Interestingly, Western blot analysis indicated that growth-associated protein 43 (GAP-43) had increased expression levels in the FVC group compared with the autograft group. This result suggested that by activating the Janus activated kinase2 (JAK2)/Phosphorylation ofsignal transducer and activator of transcription-3 (STAT3) signaling pathway, FVC upregulated Phosphorylation of signal transducer and activator of transcription-3 (P-STAT3) in vivo, resulting in the secretion of GAP-43. Collectively, a natural NGC FVC was fabricated based on FV without chemical modifications. The morphology, physical properties, cellular biocompatibility, antibacterial properties, and nerve regeneration capacity of FVC provide new insights for its further optimization and application in the field of nerve tissue engineering.

Comprehensive strategy of conduit guidance combined with VEGF producing Schwann cells accelerates peripheral nerve repair.

Peripheral nerve regeneration requires stepwise and well-organized establishment of microenvironment. Since local delivery of VEGF-A in peripheral nerve repair is expected to promote angiogenesis in the microenvironment and Schwann cells (SCs) play critical role in nerve repair, combination of VEGF and Schwann cells may lead to efficient peripheral nerve regeneration. VEGF-A overexpressing Schwann cells were established and loaded into the inner wall of hydroxyethyl cellulose/soy protein isolate/polyaniline sponge (HSPS) conduits. When HSPS is mechanically distorted, it still has high durability of strain strength, thus, can accommodate unexpected strain of nerve tissues in motion. A 10 mm nerve defect rat model was used to test the repair performance of the HSPS-SC (VEGF) conduits, meanwhile the HSPS, HSPS-SC, HSPS-VEGF conduits and autografts were worked as controls. The immunofluorescent co-staining of GFP/VEGF-A, Ki67 and MBP showed that the VEGF-A overexpressing Schwann cells could promote the proliferation, migration and differentiation of Schwann cells as the VEGF-A was secreted from the VEGF-A overexpressing Schwann cells. The nerve repair performance of the multifunctional and flexible conduits was examined though rat behavioristics, electrophysiology, nerve innervation to gastrocnemius muscle (GM), toluidine blue (TB) staining, transmission electron microscopy (TEM) and NF200/S100 double staining in the regenerated nerve. The results displayed that the effects on the repair of peripheral nerves in HSPS-SC (VEGF) group was the best among the conduits groups and closed to autografts. HSPS-SC (VEGF) group exhibited notably increased CD31+ endothelial cells and activation of VEGFR2/ERK signaling pathway in the regenerated nerve tissues, which probably contributed to the improved nerve regeneration. Altogether, the comprehensive strategy including VEGF overexpressing Schwann cells-mediated and HSPS conduit-guided peripheral nerve repair provides a new avenue for nerve tissue engineering.

Mesenchymal stromal cells for sepsis and septic shock: Lessons for treatment of COVID-19.

Sepsis is defined as life-threatening organ dysfunction caused by a deregulated immune host response to infection. The emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted this multifactorial and complex syndrome. The absence of specific treatment neither against SARS-CoV-2 nor against acute respiratory distress syndrome (ARDS), the most serious stage of this infection, has emphasized the need to find alternative treatments. Several therapeutics are currently being tested, including mesenchymal stromal cells. These cells, already used in preclinical models of ARDS, sepsis, and septic shock and also in a few clinical trials, appear well-tolerated and promising, but many questions remain unanswered.

Brain Derived Neurotrophic Factor and Glial Cell Line-Derived Neurotrophic Factor-Transfected Bone Mesenchymal Stem Cells for the Repair of Periphery Nerve Injury.

Peripheral nerve injury is a common clinical neurological disease. In our previous study, highly oriented poly (L-lactic acid) (PLLA)/soy protein isolate (SPI) nanofiber nerve conduits were constructed and exhibited a certain repair capacity for peripheral nerve injury. In order to further improve their nerve repairing efficiency, the bone mesenchymal stem cells (BMSCs) overexpressing brain derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) were introduced into the conduits as seed cells and then were used to repair the 10-mm sciatic nerve defects in rats. The nerve repair efficiency of the functional nerve conduits was evaluated by gait experiment, electrophysiological test, and a series of assays such as hemotoxylin-eosin (HE) staining, immunofluorescence staining, toluidine blue (TB) staining, transmission electron microscopy (TEM) observation of regenerated nerve and Masson's trichrome staining of gastrocnemius muscle. The results showed that the conduits containing BMSCs overexpressing BDNF and GDNF double-factors group had better nerve repairing efficiency than blank BMSCs and single BDNF or GDNF factor groups, and superior to autografts group in some aspects. These data demonstrated that BDNF and GDNF produced by BMSCs could synergistically promote peripheral nerve repair. This study shed a new light on the conduits and stem cells-based peripheral nerve repair.

Are the Immune Properties of Mesenchymal Stem Cells from Wharton's Jelly Maintained during Chondrogenic Differentiation?

BACKGROUND: Umbilical mesenchymal stem/stromal cells (MSCs), and especially those derived from Wharton's jelly (WJ), are a promising engineering tool for tissue repair in an allogeneic context. This is due to their differentiation capacity and immunological properties, like their immunomodulatory potential and paracrine activity. Hence, these cells may be considered an Advanced Therapy Medicinal Product (ATMP). The purpose of this work was to differentiate MSCs from WJ (WJ-MSCs) into chondrocytes using a scaffold and to evaluate, in vitro, the immunomodulatory capacities of WJ-MSCs in an allogeneic and inflammatory context, mimicked by IFN-γ and TNF-α priming during the chondrogenic differentiation. METHODS: Scaffolds were made from hydrogel composed by alginate enriched in hyaluronic acid (Alg/HA). Chondrogenic differentiation, immunological function, phenotype expression, but also secreted soluble factors were the different parameters followed during 28 days of culture. RESULTS: During chondrocyte differentiation, even in an allogeneic context, WJ-MSCs remained unable to establish the immunological synapse or to induce T cell alloproliferation. Moreover, interestingly, paracrine activity and functional immunomodulation were maintained during cell differentiation. CONCLUSION: These results show that WJ-MSCs remained hypoimmunogenic and retained immunomodulatory properties even when they had undergone chondrocyte differentiation.

An Ergonomic Assessment Of Four Different Donor Nephrectomy Approaches For The Surgeons And Their Assistants.

INTRODUCTION: Open surgery is increasingly being replaced by laparoscopic approaches that are more demanding for the surgical team. The physical and mental workload of these approaches have not been quantified. MATERIALS AND METHODS: A multicenter prospective study was performed evaluating the physical and mental stresses of 4 surgical approaches (open surgery [OS], standard laparoscopy [SL], hand-assisted laparoscopy [HAL], and robot-assisted laparoscopy [RAL]) for donor nephrectomy for the surgeon and their assistant. The Borg Scale was used to evaluate exertion in different body parts every 30 mins during surgery and the NASA-TLX score was used to evaluate overall workload. RESULTS: 264 nephrectomies were performed over a 33-month period and 258 questionnaires evaluating these surgeries were obtained. Surgeons experienced less left shoulder and arm exertion and left forearm and hand exertion, but greater lower back exertion, as measured by the Borg scale, with RAL. Leg exertion was significantly greater with OS. Assistant surgeons experienced increased exertion in the back, right shoulder and arm, and right forearm and hand with RAL. NASA Task load index (TLX) surgeon scores showed mental demand was similar for all 4 surgical approaches. Physical demand was lower and overall performance was higher with RAL. DISCUSSION: Four different nephrectomy surgical approaches were evaluated in a multicenter setting. Surgeon and assistant scores of physical exertions were generally in the "easy" range but confirmed that robotic surgery is an ergonomic progress compared to other techniques, except for the axial skeleton. Further, it degrades the working conditions for the assistant.

Accelerated skin wound healing by soy protein isolate-modified hydroxypropyl chitosan composite films.

In this study, a series of hydroxypropyl chitosan (HPCS)/soy protein isolate (SPI) composite films (HCSFs) with different SPI contents were developed via crosslinking, solution casting, and evaporation process. Effects of the SPI content on the structure and physical properties of the HCSFs were characterized by Fourier transform infrared spectroscopy, X-ray diffraction patterns, scanning electron microscopy, swelling kinetics analysis, and mechanical testing. The HCSFs exhibited a lower swelling ratio with an increase in the SPI content. The tensile strength was in a tunable range from 7.88 ±â€¯3.08 to 40.44 ±â€¯2.31 MPa by adjusting the SPI content. Cytocompatibility and hemocompatibility of the HCSFs were evaluated by a series of in vitro assays, including MTT assay, live/dead assay, cell morphology observation, hemolysis ratio testing, and plasma recalcification time measurement. Results showed that the HCSFs support L929 cells attachment and proliferation without obvious hemolysis, indicating good cytocompatibility and hemocompatibility. The potential of resultant HCSFs as the wound dressings was investigated using a full-thickness skin wound model in rats. Results exhibited that the HCSFs with 50% SPI content had the fastest healing speed and the best skin regeneration efficiency and may be a potential candidate as the wound dressing.

Electrodeposition to construct mechanically robust chitosan-based multi-channel conduits.

A series of electrodeposited chitosan-based multi-channel conduits (ECMC) with potential for peripheral nerve tissue engineering were constructed using a novel electrodeposition method combined with homemade molds. The structural and mechanical properties of the ECMC were characterized by scanning electron microscopy, Fourier-transformed infrared spectroscopy, X-ray diffraction patterns and mechanical testing. The results showed that the electrodeposition process did not change the chemical structure of the chitosan molecules, but endowed the ECMC with high levels of flexibility and elasticity. Hemocompatibility and cytocompatibility of the ECMC were evaluated by hemolysis assay, MTT assay and live/dead assay. The results indicated that the ECMC had a low hemolysis rate, and can promote cell proliferation and support cell adhesion. This work provides a safe and feasible electrodeposition method to construct chitosan-based conduits with potential applications for peripheral nerve tissue engineering.

Mechanical stimulations on human bone marrow mesenchymal stem cells enhance cells differentiation in a three-dimensional layered scaffold.

Scaffolds laden with stem cells are a promising approach for articular cartilage repair. Investigations have shown that implantation of artificial matrices, growth factors or chondrocytes can stimulate cartilage formation, but no existing strategies apply mechanical stimulation on stratified scaffolds to mimic the cartilage environment. The purpose of this study was to adapt a spraying method for stratified cartilage engineering and to stimulate the biosubstitute. Human mesenchymal stem cells from bone marrow were seeded in an alginate (Alg)/hyaluronic acid (HA) or Alg/hydroxyapatite (Hap) gel to direct cartilage and hypertrophic cartilage/subchondral bone differentiation, respectively, in different layers within a single scaffold. Homogeneous or composite stratified scaffolds were cultured for 28 days and cell viability and differentiation were assessed. The heterogeneous scaffold was stimulated daily. The mechanical behaviour of the stratified scaffolds were investigated by plane-strain compression tests. Results showed that the spraying process did not affect cell viability. Moreover, cell differentiation driven by the microenvironment was increased with loading: in the layer with Alg/HA, a specific extracellular matrix of cartilage, composed of glycosaminoglycans and type II collagen was observed, and in the Alg/Hap layer more collagen X was detected. Hap seemed to drive cells to a hypertrophic chondrocytic phenotype and increased mechanical resistance of the scaffold. In conclusion, mechanical stimulations will allow for the production of a stratified biosubstitute, laden with human mesenchymal stem cells from bone marrow, which is capable in vivo to mimic all depths of chondral defects, thanks to an efficient combination of stem cells, biomaterial compositions and mechanical loading.

Umbilical cord-derived mesenchymal stromal cells: predictive obstetric factors for cell proliferation and chondrogenic differentiation.

BACKGROUND: The umbilical cord is becoming a notable alternative to bone marrow (BM) as a source of mesenchymal stromal cells (MSC). Although age-dependent variations in BM-MSC are well described, less data are available for MSC isolated from Wharton's jelly (WJ-MSC). We initiated a study to identify whether obstetric factors influenced MSC properties. We aimed to evaluate the correlation between a large number of obstetric factors collected during pregnancy and until peripartum (related to the mother, the labor and delivery, and the newborn) with WJ-MSC proliferation and chondrogenic differentiation parameters. METHODS: Correlations were made between 27 obstetric factors and 8 biological indicators including doubling time at passage (P)1 and P2, the percentage of proteoglycans and collagens, and the relative transcriptional expression of Sox-9, aggrecans, and total type 2 collagen (Coll2T). RESULTS: Amongst the obstetric factors considered, birth weight, the number of amenorrhea weeks, placental weight, normal pregnancy, and the absence of preeclampsia were identified as relevant factors for cell expansion, using multivariate linear regression analysis. Since all the above parameters are related to term, we concluded that WJ-MSC from healthy, full-term infants exhibit greater proliferation capacity. As for chondrogenesis, we also observed that obstetric factors influencing proliferation seemed beneficial, with no negative impact on MSC differentiation. CONCLUSIONS: Awareness of obstetric factors influencing the proliferation and/or differentiation of WJ-MSC will make it possible to define criteria for collecting optimal umbilical cords with the aim of decreasing the variability of WJ-MSC batches produced for clinical use in cell and tissue engineering.

Mechanobiology of mesenchymal stem cells: Which interest for cell-based treatment?

Thanks to their immune properties, the mesenchymal stem cells (MSC) are a promising source for cell therapy. Current clinical trials show that MSC administrated to patients can treat different diseases (graft-versus-host disease (GVHD), liver cirrhosis, systemic lupus, erythematosus, rheumatoid arthritis, type I diabetes…). In this case, the most common mode of cell administration is the intravenous injection, and the hemodynamic environment of cells induced by blood circulation could interfere on their behavior during the migration and homing towards the injured site. After a brief review of the mechanobiology concept, this paper will help in understanding how the mechanical environment could interact with MSC behavior once they are injected to patient in cell-based treatment.

Comparison of MSC properties in two different hydrogels. Impact of mechanical properties.

Once articular cartilage is damaged, it has poor ability to heal. At present, alginate-based hydrogels have 3D-dimensional physical structures with great potential for applications in carilage tissue engineering. For osteochondral defect, it will be necessary to use stratified scaffold to mimic zonal organization of cartilage. This study aims to compare the characteristics of alginate (Alg)/hyaluronic acid (HA) hydrogels which will mimic cartilage with alginate (Alg)/hydroxyapatite (Hap) hydrogels which will mimic subchondral bone. In this work, we fabricated the 3D-Alg/HA and Alg/Hap hydrogel scaffolds by the original spraying method. From the physical-mechanical properties, we compared mechanical behaviour of Alg/HA and Alg/Hap hydrogel scaffolds, which were examined using indentation testing and viscosity behaviour. This results showed that the Alg/Hap hydrogels exhibited a relative high mechanical strength, as well as the viscosity of Alg/Hap hydrogels is slight slower than Alg/HA hydrogels. However, autoclaving has more deleterious effect on the mechanical and viscosity properties of Alg/HA and Alg/Hap hydrogels. Cytotoxicity was evaluated through the culture of hydrogel beads-laden Wharton's jelly mesenchymal stem cells (WJ-MSC). In addition, the chondrogenic differentiation of WJ-MSC encapsulated into Alg/HA and Alg/Hap hydrogels were performed by histological analyzing during 30 days of culture. From these results, the percentage of living cells for Alg/Hap is significantly higher than Alg/HA, which also is associated with the results of shear viscosity. Both of hydrogels exhibited differentiate into chondrocyte matrix as collagen and proteoglycans. In conclusion, Alg/Hap hydrogels presented better mechanical property, cytocompatibility and differentiation characteristics than Alg/HA hydrogels.

Effect of nicotine on the proliferation and chondrogenic differentiation of the human Wharton's jelly mesenchymal stem cells.

BACKGROUND: Osteoarthritis (OA) is a chronic joint disease characterized by a progressive and irreversible degeneration of articular cartilage. Among the environmental risk factors of OA, tobacco consumption features prominently, although, there is a great controversy regarding the role of tobacco smoking in OA development. Among the numerous chemicals present in cigarette smoke, nicotine is one of the most physiologically active molecules. OBJECTIVE: The aim of the study was (i) to measure the impact of nicotine on the proliferation and chondrogenic differentiation of mesenchymal stem cells from the human Wharton's jelly (hWJ-MSCs) into chondrocytes, (ii) to investigate whether the α7 nicotinic acetylcholine receptors (nAChRs) was expressed in hWJ-MSCs and could play a role in the process. The project benefits from the availability of an umbilical cord bank from which hWJ-MSCs were originated. METHODS: The hWJ-MSCs were cultured and used up to passage 5. The proliferation of hWJ-MSCs with 5 µM nicotine was measured by the MTT assay on the 1st, 2nd, 3rd, and 6th day. Flow cytometry analysis was used to detect cell apoptosis/necrosis by Annexin V/PI double-staining. The chondrogenic differentiation grade of hWJ-MSCs induced by TGFß3 was assessed by the Sirius red and Alcian blue staining. The expression of markers genes was followed by quantitative real-time PCR. The expression of nAChRs was followed by RT-PCR. The functional activity of α7 nAChR was evaluated by calcium (Ca2+) influx mediated by nicotine using the Fluo-4 NW Calcium assay. RESULTS: The proliferation of hWJ-MSCs was significantly impaired by nicotine (5 µM) from the 3rd day of treatment, but nicotine did not significantly induce modifications on the viability of hWJ-MSCs. Alcian blue staining indicated that the amount of proteoglycan was more abundant in control group than in the nicotine group, but no difference was observed on the total collagen amount using Sirius red staining. The mRNA expression of Sox9, type II collagen (Col2a1), aggrecan in control group was higher than in the nicotine group. We found that hWJ-MSCs expressed α7 nAChR. The receptor agonist nicotine caused calcium (Ca2+) influx into hWJ-MSCs suggesting that the calcium ion channel α7 homopolymer could mediate this response. CONCLUSIONS: At the concentration used, nicotine had an adverse effect on the proliferation and chondrogenic differentiation of hWJ-MSCs which was probably impaired through a α7 nAChR mediation.

Is there a cause-and-effect relationship between physicochemical properties and cell behavior of alginate-based hydrogel obtained after sterilization?

Alginate-based hydrogel scaffolds are widely used in the field of cartilage regeneration and repair. If the effect of autoclaving on the alginate powder is well known, it is not the same for the possible effects of the sterilization UV treatment on the properties of the hydrogel after polymerization. To select an effective sterilization treatment of alginate-based materials, one must find what are inter-relationship between the characteristics (chemical, physical and mechanical) of alginate-based hydrogel during sterilization, and what consequences have affected on cell behavior. In this study, we investigated the influence of UV sterilization treatments (UV-1 and UV-2: 25 and 50min, respectively) and autoclaving to obtain alginate (Alg)/hyaluronic acid (HA) hydrogel, as well as further evaluated the relationship between physicochemical properties and cell behavior of Alg/HA hydrogel after UVs and autoclaving. The physicochemical properties of this mixture at the powder or polymerized states were analyzed using ATR-FTIR, HPLC-SEC, rheological, indentation testing and sterility testing. The cell behaviors of hydrogels were evaluated by cell viability and proliferation, and chondrogenic differentiation. The effects of treatment parameters and their correlation with the others characteristics were determined statistically by Principal Component Analysis (PCA). In this study, we have shown that the cell behavior in alginate-based hydrogels was not only regulated by physicochemical properties (as molar mass or/and viscosity), but also associated with the controlling of sterilization time. It can provide a basis for choosing an effective method of sterilization, which can keep the mechanical or physical-chemical properties of Alg-based hydrogel scaffold and maintain its cytocompatibility and its ability to induce chondrogenesis from mesenchymal stem cells.

Improvement of functionality after chitosan-modified zein biocomposites.

A series of chitosan-modified zein composite films were fabricated from zein and chitosan by a process involving blending, solution casting and evaporation. Effects of chitosan content on the structure and physical properties of the composite films were investigated by Fourier transform infrared spectroscopy, differential scanning calorimetry, scanning electron microscopy, tensile testing, water absorption measurement and water contact angle measurement. The results showed that the zein/chitosan composite films were fabricated successfully due to the formation of hydrogen bonds between zein and chitosan, and the thermal stability, water absorption, hydrophilicity, tensile strength, flexibility of the composite films increased with an increase in chitosan content from 0 to 50%. The cytotoxicity and cytocompatibility of the composite films were evaluated by 3-[45-dimethyl-2-thiazoly1]-25-diphenyl-2H-tetrazolium bromide (MTT) assay and in vitro cell culture, which showed that the films have non- or low-cytotoxicity, and chitosan promoted the growth, adhesion and proliferation of the cells. These results indicated that chitosan-modified zein composite films might have potentials applications as biomaterials.