Identification and expression assay of calcium-dependent protein kinase family genes in Hevea brasiliensis and determination of HbCDPK5 functions in disease resistance.

Calcium (Ca2+) signaling is one of the earliest factors to coordinate plant adaptive responses. As direct sensors and activators of Ca2+ signals, calcium-dependent protein kinases (CDPKs) were reported to be widely involved in regulating different biotic and abiotic stress stimuli. In this study, 32 Hevea brasiliensis CDPK (HbCDPK) genes were predicted and classified into four subgroups. Among them, the full-length coding sequences of 28 HbCDPK genes were confirmed by RT-PCR and verified by sequencing. Putative cis-elements assay in the promoters of HbCDPKs showed that most of the HbCDPK genes contained gibberellic acid-responsive element (GARE), abscisic acid-responsive element (ABRE), salicylic acid-responsive element (SARE), defense and stress responsive element (TC-rich repeats) and low-temperature response element (LTR), which could be activated by different biotic and abiotic stresses. Real-time PCR analysis indicated that 28 HbCDPK genes respond to infection of pathogenic fungi and a variety of phytohormones. Subcellular localization was observed with most HbCDPKs located in cell membrane, cytoplasm or organelles. Some HbCDPKs were confirmed to cause reactive oxygen species (ROS) production and accumulation in rubber tree mesophyll protoplast directly. HbCDPK5 was strongly induced by the inoculation with Colletotrichum gloeosporioides and was chosen for further analysis. HbCDPK5 localized to the cell membrane and cytoplasm, and obviously regulated the accumulation of ROS in rubber tree mesophyll protoplast. Overexpression of HbCDPK5 in Arabidopsis enhanced the resistance to Botrytis cinerea. These results indicate that rubber tree CDPK genes play important roles in plant disease resistance.

Comparative Physiological Analysis of Methyl Jasmonate in the Delay of Postharvest Physiological Deterioration and Cell Oxidative Damage in Cassava.

The short postharvest life of cassava is mainly due to its rapid postharvest physiological deterioration (PPD) and cell oxidative damage, however, how to effectively control this remains elusive. In this study, South China 5 cassava slices were sprayed with water and methyl jasmonate (MeJA) to study the effects of MeJA on reactive oxygen species, antioxidant enzymes, quality, endogenous hormone levels, and melatonin biosynthesis genes. We found that exogenous MeJA could delay the deterioration rate for at least 36 h and alleviate cell oxidative damage through activation of superoxide dismutase, catalase, and peroxidase. Moreover, MeJA increased the concentrations of melatonin and gibberellin during PPD, which had a significant effect on regulating PPD. Notably, exogenous MeJA had a significant effect on maintaining cassava quality, as evidenced by increased ascorbic acid content and carotenoid content. Taken together, MeJA treatment is an effective and promising way to maintain a long postharvest life, alleviate cell oxidative damage, and regulate storage quality in cassava.

Overexpressing OsMAPK12-1 inhibits plant growth and enhances resistance to bacterial disease in rice.

Mitogen-activated protein kinases (MAPKs) play important roles in plant growth and development, plant abiotic stresses signalling pathway and plant-pathogen interactions. However, little is known about the roles of MAPKs in modulating plant growth and pathogen resistance. In this study, we found that OsMAPK12-1, an alternatively spliced form of BWMK1 in rice (Oryza sativa L.), was induced by various elicitors, such as jasmonic acid, salicylic acid, melatonin and bacterial pathogens. To further investigate the involvement of OsMAPK12-1 in plant growth and stress responses to bacterial pathogens, we constructed OsMAPK12-1 overexpression and knockdown (RNAi) transgenic rice lines. Interestingly, overexpressing OsMAP12-1 inhibited seed germination and seedling growth. Additionally, the OsMAP12-1-overexpression lines displayed enhanced disease resistance against Xanthomonas oryzae pv. oryzae PXO99 and Xanthomonas oryzae pv. oryzicola RS105, whereas the OsMAPK12-1-RNAi lines were more susceptible to these pathogens than wild type. These results suggest that OsMAPK12-1 plays a negative role in plant growth and positively modulates disease resistance against bacterial blight and streak in rice.

[The three-dimensional culture of adult mesenchymal stem cells for intervertebral disc tissue engineering].

Intervertebral disc (IVD) degeneration is one of the major causes of low back pain. As current clinical treatments are aimed at restoring biomechanical function and providing symptomatic relief, the methods focused on biological repair have aroused interest and several tissue engineering approaches using different cell types have been proposed. Owing to the unsuitable nature of degenerate cells for tissue engineering, attention has been given to the use of mesenchymal stem cells (MSCs). In this connection, we have made a study on the characteristics of MSCs derived from adult bone marrow and on the feasibility of constructing IVD tissue-engineering cell under a Three-Dimensional Pellet Culture System. The human bone marrow MSCs were isolated and purified with density gradient solution and attachment-independent culture system. MSCs isolated using this method are a homogeneous population as indicated by morphology and other criteria. They have the capacity for self-renewal and proliferation, and the multilineage potential to differentiate.

Histological study of surface modified three dimensional poly (D, L-lactic acid) scaffolds with chitosan in vivo.

Biocompatibility and tissue regenerating capacity are essential for biomaterials that used in tissue engineering. The aim of this study was to histologically assess the tissue reactions and bone conductivities of surface modified three dimensional (3-D) poly (D, L-lactic acid) (PDLLA) scaffolds, which were coated with chitosan via a physical entrapment method. The native PDLLA scaffold was prepared via thermally induced phrase separation technique and was characterized by scanning electron microscopy (SEM) and differential scanning calorimetry (DSC). Osteocalcin assay, a method to evaluate the bone formation potential, has shown that the osteocalcin production in chitosan-modified 3-D PDLLA scaffold group was significantly higher (p < 0.05) than that of in control. The tissue reactions and bone conductivities between surface modified PDLLA and native PDLLA scaffolds were evaluated using a rabbit radialis defect model in vivo and compared at different implantation intervals (2, 4, 8 and 12 weeks). The histological results have shown a higher bone formation potential and better biocompatibility of chitosan-modified 3-D PDLLA scaffolds as compared with the control group scaffolds.

Surface modification of three-dimensional poly(d,l-lactic acid) scaffolds with baicalin: a histological study.

To improve the biocompatibility of three dimensional (3D) poly(d,l-lactic acid) (PDLLA), surface modification with baicalin was performed via a physical entrapment method in this study. The tissue reactions and bone conductivities of such modified PDLLA scaffolds were histologically assessed by using a rabbit radialis defect model in vivo. The native PDLLA scaffolds were prepared via a thermally induced phase separation technique and were characterized by scanning electron microscopy and fluorescence microscopy. The tissue reactions and bone conductivities of both baicalin-modified PDLLA scaffolds and native PDLLA scaffolds were comparably evaluated with histological assay and histomorphometry at different implantation intervals (2, 4, 8 and 12 weeks). Osteocalcin assay, a method to evaluate the bone formation potential, has shown that the osteocalcin production in the baicalin-modified 3D PDLLA scaffold group was significantly higher (p<0.01 or <0.05) than that in the control. Histological observation and histomorphometry results reflected a higher bone formation potential and better biocompatibility of baicalin-modified PDLLA scaffold when compared with those of the native PDLLA scaffolds.

[Use of cortical bone plates allografts for reconstruction of femoral fracture: an experimental study in goats].

OBJECTIVE: To study the outcome of reconstructing goat femoral fracture by use of cortical bone plates allografts that have been kept by deep-freezing at -70 degrees C for 4 weeks after being treated with 48 degrees C ethylene oxide. METHODS: The recipients, sixteen 10-12-month-old goats with fractures of right femur were subjected the operation for transplanting the cortical bone plates allografts in the medial, lateral and back sides of fractured femurs. The goats were sacrificed and the specimens were procured at 3, 6, 12, and 24 weeks after surgery for X-ray photography, Chinese ink perfusion, tetracycline fluorescence labeling and histological observation in order evaluate the healing of fracture and the incorporation of cortical bone plates allografts. RESULTS: The allograft strut was found revascularized at 6 weeks after surgery in the fracture group, whereas at 3 weeks in the control group. The tetracycline fluorescence labeling was poor in the fracture group as compared with that in the control group from 3 weeks to 6 weeks, but it was better in the fracture group than in the control group beyond 6 weeks after surgery. Fracture was healed and bone conjunction between allograft strut and host bone was seen at weeks after operation. The allograft strut was incorporated in host bone, the ability of remodeling of allograft strut and the size of femoral cortex were better in the fracture group than in the control group at 24 weeks after surgery. The fracture was displaced in 19% animals and the allograft bone plates were not fractured. CONCLUSION: The use of allograft strut pre-treated by ethylene oxide sterilization and deep-freezing could underpin fixation and promote healing of femoral fracture, and it can increase bone reservation and augment the strength of femur once the allograft strut is incorporated in the host bone.

Effects of baicalin-modified poly(D,L-lactic acid) surface on the behavior of osteoblasts.

In the present study, the functions of rat calvaria osteoblasts on baicalin-modified poly(D,L-lactic acid) (PDLLA) films were investigated in vitro. The surface characteristics of surfaces (both modified and control) were investigated by water contact angle measurement and electron spectroscopy for chemical analysis (ESCA). Cell morphologies on these surfaces were examined by scanning electron microscopy (SEM). Cell adhesion and proliferation were used to assess cell growth on the modified and control surfaces. The MTT assay was used to determine cell viability and alkaline phosphatase (ALP) activity was performed to evaluate differentiated cell function. Compared to control films, cell attachment of osteoblasts on baicalin-modified PDLLA film was significantly higher (P<0.05 and P<0.01) after 6 h and 8 h culture, and cell proliferation was also significantly greater (P<0.05 and P<0.01) at the end of 4th and 7th day, respectively. The MTT assay suggested that the cell viability of osteoblasts cultured on baicalin-modified PDLLA film was significantly higher (P<0.05) than that seeded on the control. Meanwhile, the ALP activity of osteoblasts cultured on modified films was also considerably enhanced (P<0.01) compared to that found on control. These results revealed that the biocompatibility PDLLA could be improved by surface modification with baicalin.

Improvement of the functions of osteoblasts seeded on modified poly(D,L-lactic acid) with poly(aspartic acid).

One of the challenges in the field of tissue engineering is the development of biomaterial/cell interactions. For the purposes of the present study, two molecular weights of poly(aspartic acid) (PASP) were used to modify poly(D,L-lactic acid) (PDLLA) films in order to enhance their cell affinity. The properties of the PDLLA-modified surfaces and the controls were investigated by water contact angle measurement and electron spectroscopy for chemical analysis (ESCA). These data reflect the change in the biocompatibility of modified PDLLA surfaces. Then rat osteoblasts were seeded onto these modified surfaces and on controls to examine their effects on cell adhesion and proliferation. Cell morphologies on these surfaces were studied by scanning electron microscopy (SEM), and cell viability was evaluated with a MTT assay. In addition, differentiated cell function was assessed by measuring alkaline phosphatase (ALP) activity. The results suggest that PASP-modified surfaces may enhance the interactions between osteoblasts and PDLLA films.

Modulation of osteoblast function using poly(D,L-lactic acid) surfaces modified with alkylation derivative of chitosan.

Poly(D,L-lactic acid) (PDLLA) was modified with alkylated chitosan (N-butyl chitosan and N-cetyl chitosan), and the effects of modified films on the functions of rat osteoblasts were investigated. The characteristics of surfaces (both modified and control) were examined by water contact angle measurement and electron spectroscopy for chemical analysis (ESCA). Cell morphologies on these surfaces were taken using scanning electron microscopy (SEM). Cell attachment and proliferation were used to assess cell behavior on modified surface and control. MTT assay was used to determined cell viability, and alkaline phosphatase (ALP) activity was taken to evaluate differentiated cell function. Compared with the untreated films, no significant difference in cell attachment of osteoblasts was found on the modified films at a period of 8 h (p > 0.05). However, cell proliferation of N-butyl chitosan rather than N-cetyl chitosan modified PDLLA films was significantly higher than that found on control one (p < 0.05) at the end of the 4th and 7th days. The cell viability of osteoblasts on N-butyl chitosan modified PDLLA films were found higher than that on control (p < 0.05). These results suggested that N-butyl chitosan contributed greater than N-cetyl chitosan when used to modify PDLLA films for improving its biocompatibility.

[A tissue-engineered strain scaffold for three-dimensional cell cultures].

This article introduces a three-dimensional scaffold which is used to perform three-dimensional cell culture under mechanical stretch from the point of construction of tissue-engineered tissue. The composition, structure, surface characteristics, mechanical property, and cell compatibility of the scaffold have been studied by using surface chemistry and material mechanics testing methods. The results indicate that the polyvinyl alcohol (PVA) sponge, which is water-tolerant, coated with Poly-DL-lactic-co-glycolic acid (PLGA) possesses a good nature in appropriate surface feature, porosity, elastic recoil, and cell compatibility. These features provide wide options for using this scaffold to study the effects of mechanical stretch on cells maintained in three-dimensional culture to provide a three-dimensional matrix.

Influence of different surface modification treatments on poly(D,L-lactic acid) with silk fibroin and their effects on the culture of osteoblast in vitro.

The objective of this study was to investigate the efficiency of two treatments for poly(D,L-lactic acid) (PDLLA) surface modification using silk fibroin. one chemical treatment and one physical treatment: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (WSC) and entrapment. The properties of control films, WSC-modified and entrapment-treated PDLLA films were investigated by water contact angle measurement and electron spectroscopy for chemical analysis (ESCA). The water-contact angle measurement indicated the change of hydrophilicity and the ESCA analysis suggested that the modified PDLLA film using silk fibroin became enriched with nitrogen atoms. The biocompatibility of PDLLA film might be altered, which in turn would affect the functions of cells that were seeded on it. Therefore, attachment and proliferation of osteoblasts that were seeded on modified PDLLA films and control films were examined. Cell viability was evaluated by the MTT assay and differentiated cell function was assessed by measuring alkaline phosphatase activity. These results suggested that silk fibroin was used to modify PDLLA surface via WSC and that entrapment could improve the interactions between osteoblasts and PDLLA films. The entrapment treatment was more effective thin WSC treatment to accomplish the goal of surface modification.

Surface modification of poly (D,L-lactic acid) with chitosan and its effects on the culture of osteoblasts in vitro.

Chitosan is a good biodegradable natural polymer, widely used in biomedical fields. In this study, chitosan was used to modify the surface of poly (D,L-lactic acid) (PDLLA) in order to enhance its cell affinity. The properties of a modified PDLLA surface and control were investigated by contact angle and electron spectroscopy for chemical analysis (ESCA), which indicated the changes in surface energy and chemical structure. Scanning electron microscopy (SEM) observation displayed differences in surface morphology between the chitosan-modified film and the control. These data reflected that PDLLA films could be modified with chitosan and in turn may affect the biocompatibility of the modified films. Therefore, adhesion and growth of osteoblasts on modified PDLLA films as well as control were studied. Cell morphologies on the films were examined by SEM and cell viability was evaluated using an MTT assay; the differentiated cell function was assessed by measuring alkaline phosphatase (ALP) activity. The ALP activity of modified PDLLA films was significantly higher than that found on the control (p < 0.01). The proliferation of osteoblasts on modified films was also found to be higher than that on the control (p < 0.05), suggesting that chitosan could be used to modify PDLLA and then enhance its cell biocompatibility.

Poly(D,L-lactic acid) surfaces modified by silk fibroin: effects on the culture of osteoblast in vitro.

The objective of this study was to modify the surface of poly(D,L-lactic acid) (PDLLA) with different molecular weight of silk fibroins, and assess the effects of the modified surfaces on the functions of rat osteoblasts cultured in vitro. The properties of the modified PDLLA surface and the control one were investigated by contact angle and electron spectroscopy for chemical analysis (ESCA). The former indicated the variation of hydrophilicity and the latter suggested that the modified PDLLA film using silk fibroin is enriched with nitrogen atoms. The biocompatibility of the PDLLA film may be altered and in turn affects the seeded cell functions. Therefore, attachment and proliferation of osteoblasts seeded on the modified PDLLA films and the control one were examined. Cell morphologies on these films were studied by scanning electron microscopy (SEM) and cell viability was evaluated by MTT assay. In addition, differentiated cell function was assessed by measuring the alkaline phosphatase (ALP) activity. These results suggest that the silk fibroin-modified PDLLA surface can improve the interaction between osteoblasts and the PDLLA films.

[Initial study on three-dimensional culture of tenocytes under cyclic mechanical stretch].

OBJECTIVE: To detect the effect of mechanical stretch on shape, alignment, proliferation, and metabolism of tendon cells maintained in three-dimensional culture. METHODS: A cyclic mechanical strain apparatus for three-dimensional cell cultures was developed. Based on the apparatus, a specific stretch pattern (10% elongation, 12 stretches/min for 15 min of each hour) was applied to tenocytes-scaffolding composites. RESULTS: Initial studies demonstrated that the stretch-mediated effects on cell division, DNA synthesis, and metabolism in such cultures were influenced by the amplitude, frequency, periodicity, and duration of the applied stretch. After 48 hours' exposure to the stretch, the cell number and [3H] thymidine incorporation into DNA were increased, compared with those of the nonstretched controls(P < 0.05). Under the stretch pattern, the shape of cells changed to oblate and spread to the direction of the stretch. The cyclic stretch also caused an increase in collagen synthesis by tendon cells (P < 0.05), which was predominant in type I. CONCLUSION: Cyclic mechanical stretches act directly to stimulate tendon cell growth and these results are compatible with a significant role for stretch in tissue-engineered tendon construction.