The modification research of bionic silk fibroin nerve guidance conduits by silk sericin / 中华显微外科杂志

Objective To explore the modification of bionic silk fibroin nerve conduits (SF-NCs) by silk sencin.Methods The innovative SS/SF blended-NCs was fabricated by a vertical sequential cooling thermal induced phase separation (TIPS) processing with SF solution added sericin in proportion,its morphology was observed by Scanning electron microscopy (SEM),X-ray diffraction (XRD) and infrared spectroscopy (FTIR) were used to detect its internal molecular structure.MTT assay was used to quantitatively analyzed the PC12 cells viability co-cultured with the innovative SS/SF-NCs,SEM was used to observe the adhesion and morphology of PC12 cells seeded into the innovative SS/SF,PC12 cells were used to assess the NGF bioactivity released from the SS/SF.Results The SEM results showed that the new fabricated SS/SF-NCs had linearly oriented lamellar-like multiple-channel which distributed evenly,got great changes on the channel microstructure and their mechanical properties had been greatly improved,compared to SF-NCs.The XRD and FTIR results showed that the SS/SF-NCs had the similar internal molecular structure with natural silk.The spaces between parallel lamellar-like channels,porosities and compressive strengths of the SS/SF-NCs decreased with decreasing Sequential freezing temperature.MTT assay results showed that the viability of PC12 cell was better than the control group (P ＜ 0.05).The SEM observation indicated that PC12 cells showed good adhesion and differentiation with neuritis outgrowth during the period of co-culture with the SS/SF-NCs.NGF release from the innovative SS/SF-NCs was prolonged over 4 weeks,and remained bioactive.Conclusion The new fabricated SS/SF-NCs modified though silk sericin,which was highly bionic the structure of peripheral nerve fasciculus,had excellent mechanical properties and could be used as another alternative of artificial nerve conduits.

Preliminary fabrication of tissue engineered veins containing valves using bone marrow mesenchymal stem cells and biodegradable scaffolds in vitro / 中国组织工程研究

BACKGROUND:Chronic venous insufficiency is a major health problem worldwide. Clinical treatments include venous valve repair and venous segment containing valve transplantation. However, these are invasive procedures, and the supply of vein containing valves is limited. Significant progress in the fields of tissue engineering and regenerative medicine has been made towards the creation of tissue engineered vascular grafts for the repair of damaged or malformed vessels. It has been reported that using tissue engineering, a tissue engineered vein containing valves constructed with self-derived endothelial cells and al ogeneic acellular matrices can provide the complex physiological valve structure and mechanical stability, but this elicited an immunogenic response. OBJECTIVE:To create a viable and functional vein containing valves, which has the ability to grow, repair, and imitate natural tissues. METHODS:Bone marrow mesenchymal stem cells were obtained from Beagle dogs by density gradient centrifugation and adherence methods. Bone marrow mesenchymal stem cells were cultured in vitro. Fol owing isolation and culture the cells were examined using flow cytometry and identified by direct induction towards the osteogenic and adipogenic lineages. We fabricated biodegradable venous scaffold containing valves using the method of injection molding combined with thermal y induced phase separation. Based on the self-made cast, a three-dimensional biodegradable vein scaffold containing valves was constructed from poly(lactic-co-glycolic acid). Morphological structure was tested. Bone marrow mesenchymal stem cells were used as seed cells to be seeded onto the lumen of the tissue engineered vein scaffold containing valves in vitro and then incubated for 2 weeks. RESULTS AND CONCLUSION:Scanning electron microscopy images showed that the scaffold demonstrated sufficient porosity. Cultured cells expressed mesenchymal cellmarkers, CD44 and CD29, but did not express hematopoietic cellmarkers, CD34 and CD45 at the same time point. Scaffolds were nontoxic to cells and were favorable for the growth and migration of bone marrow mesenchymal stem cells. cells attached on the surface of poly(lactic-co-glycolic acid) scaffolds formed a confluent layer after incubation. The cellular constructs were tested in vitro, and the valve leaflets were functional y capable of opening and closing when stimulated. These results suggested that the tissue engineered vein containing valves have been successful y constructed by using a three-dimensional poly(lactic-co-glycolic acid) scaffold and bone marrow mesenchymal stem cells as seed cells. Tissue engineered vein containing valves is potential y useful for the substitution and regeneration of vein valves.

PTMC-F127-PTMC thermosensitive hydrogel as mitomycin C delivery implant for rabbit trabeculactory surgery / 中华实验眼科杂志

Background Mitomycin C (MMC) is still irreplaceable until now.While it's current administration method has proved less than effective in the treatment of refractory glaucoma.To construct a MMC sustained release system which can maintain effective concentration and reduce toxicity is important for postoperative scarring regulation after glaucoma filtration surgery.Objective To evaluate the postoperative effect of use PTMC-F127-PTMC thermosensitive hydrogel as a new drug delivery carrier to sustained release MMC in rabbit trabeculactory.Methods Sixty rabbits,aged 10 to 14 weeks,were divided into 5 trabeculectomy groups in accordance with the random number table,including surgery only group,blank PTMC-F127-PTMC group and three sustained groups with 0.1 ml PTMC-F127-PTMC loaded with 0.05,0.10,0.20 g/L MMC injected after surgery.The MMC concentration of anterior chamber aqueous in three sustained release group with 0.1 ml PTMC-F127-PTMC loaded with 0.05,0.1,0.2 g/L MMC injected after surgery were tested by Guangzhou Analysis and Testing Center using high performance liquid chromatograph-mass spectrometer (HPLC-MS).At 3,7 days postoperatively,0.1 ml aqueous humor from 2 random selected rabbits in each group was extracted using 1 ml syringe with 30G needles from corneal limbus.At postoperative 1,3,5,7,10,14,28 days,bleb width and depth were calculated with caliper measurements and height was graded semiquantitatively by slit-lamp examinations,intraocular pressure (IOP) was measured with Tonopen.And corneal endothelial cell densities were examined by corneal endothelial counting before and 28 days after surgery.Sequential sections of the operative region were prepared and stained with hematoxylin and eosin and proliferating cell nuclear antigen (PCNA) after taking off the eyeballs from dead rabbits at 28 days later.Results MMC could be sustained released from PTMC-F127-PTMC hydrogel for more than 20 days.The mean postoperative bleb survival time in trabeculectomy surgery only group,blank hydrogel group and three sustained release groups were (5.3 ± 0.4),(5.5 ± 0.4),(12.2 ± 1.0),(25.1 ± 0.9),(26.7 ± 0.7) days respectively,the difference between each group was significantly (F =123.200,P =0.000).0.05 g/L MMC sustained release group has a better bleb survival time than that of surgery only group and blank hydrogel group (P =0.000).Compared with other groups,0.10 g/L and 0.20 g/L MMC sustained group has the longest bleb survival time (P =0.000),and more obvious IOP downtrending (F=53 010.000,P＜0.01).But the difference between the two groups was not significant.There was no difference in cornea endothelia cells counts between each group and no MMC was detected in aqueous humors.Histopathology test shows that the inflammatory response and fibrosis were lighter in MMC sustained release group,with stronger proliferation inhibition.Conclusions PTMC-F127-PTMC thermosensitive hydrogel can be a new drug delivery carrier to sustained release MMC.Sustain release MMC can extent bleb survival time with low toxicity.

Adipose differentiation and adipose tissue engineering of bone marrow-derived mesenchymal stem cells using pluronic F-127 hydrogel in vitro / 生物医学工程学杂志

The aim of this study is to investigate the growth and proliferation of bone marrow mesenchymal stem cells (BMSCs) three-dimensionally cultured in Pluronic F-127 gel, in order to explore the cellular compatibility of gel and to investigate the feasibility of BMSCs differentiating into adipocytes in gel. Rat BMSCs were isolated from adult bone marrow, and then cultured and amplified in vitro. The BMSCs derived from the 4th passage were seeded on the scaffolds and incubated in adipogenic stimuli culture to differentiate into adipocytes. BMSCs were dispersed into gel and cultured in vitro for two weeks then the status of adhesion, growth and proliferation of the cells were observed. The edipogenic differentiation of the BMSCs was assessed by cellular morphology and further confirmed by Oil Red O staining. BMSCs were able to attach, grow and proliferate well in Pluronic F-127 gel. The BMSCs differentiated into adipocytes in gel in the presence of adipogenic stimuli over a period of 2 weeks. After only 4 days of adipogenic induction, small lipid droplets were observed within BMSCs in gel wells treated with differentiation media. At the end of 14 days, in the presence of differentiation media in gel, the size of the lipid droplets increased to occupy most of the cytoplasm, consistent with differentiation of BMSCs into adipocytes. Lipid droplets in differentiating BMSCs were positively stained with Oil Red O in the presence of differentiation media in the Pluronic F-127 treatment. We demostrated BMSCs incubated in the 3D Pluronic F-127 gel scaffolds could be induced and differentiated into adipocytes. The system for inducing differentiation of BMSCs into adipocytes is promising to apply in the construction of tissue engineering adipose tissue and the repair of fat injury, and Pluronic F-127 gel may be a suitable scaffold for cellular therapy of BMSCs.

Combined use of RGD-peptide modified PLGA and TGF-beta1 gene transfected MSCs to improve cell biobehaviors in vitro / 华中科技大学学报（医学）（英德文版）

In order to improve the surface properties of PLGA polymer for a better material/cell interface to modulate the cells behaviors, we prepared a novel three-block copolymer, PLGA-[ASP-PEG], and immobilized an RGD-containing peptide, Gly-Arg-Gly-Asp-Ser-Pro-Cys (GRGDSPC) on the surface of it. Transforming growth factor-beta1 (TGF-beta1) was transfected into bone marrow stromal cells (MSCs) employed as seeded cells. Cell adhesion, spreading, proliferation and differentiation on this material were investigated. The results showed that the cell adhesive ratio on RGD-modified materials was higher than on un-modified materials (P<0.05). The extent of cell spreading was also wider on RGD-modified materials than on un-modified materials. Cell proliferation indices of transfected MSCs were increased as compared with the un-transfected MSCs (P<0.05). The ALP activities in the MSCs cultured with RGD-modified materials were higher than on un-modified materials after 14 days (P<0.05), and those in transfected MSCs were higher than in un-transfected MSCs (P<0.05). It was suggested that the combined use of RGD-modification and TGF-beta gene transfection could improve the interaction of biomaterial and cells.

Cellular compatibility of poly (lactic-co-glycolic acid)scaffold / 中国组织工程研究

BACKGROUND:The poly-lactic acid and its ramifications have many advantages, such as eligible biocompatibility,nontoxicity of degradation product, easy procession and suitable intensity. Thus, they have been widely used in bone tissue engineering.OBJECTIVE: To study the cellular biocompatibility and in vitro adhesion of poly(lactic-co-glycolic acid) (PLGA) scaffold and bone marrow stromal stem cells (BMSCs) so as to provide a basis for preparation a PLGA scaffold that can load many cytokines.DESIGN: Contrast observation.SETTING: Department of Orthopaedics and Traumatology in Nanfang Hospital of Southern Medical University.MATERIALS: The experiment was carried out in Key Laboratory of Tissue Construction and Detection of Guangdong Province from September 2004 to June 2005. One New Zealand healthy male rabbit (2 months old, 1.5-2.0 kg weight)was adopted in this study. Experimental materials: PLGA scaffold was obtained from Institute of Polymer Science in Sun Yat-sen University); beta-tricalcium phosphate (β-TCP) was supplied by AO Company (Switzerland).METHODS: Bone marrow was aspirated from the New Zealand rabbit. Mononuclear cells were harvested using whole bone marrow culturing, then were induced and amplified in the conditional culture medium. BMSCs were inoculated onto the PLGA and β-TCP at a concentration of 1 ×109 L-1, while those in the medium without materials were taken as blank control group. The development of implanted cells and the adhesion between cells and materials were observed with phase contrast microscope and scanning electron microscope. The proliferation and cycle of cells were tested with MTT method and flow cytometry.MATN OUTCOME MEASURES: ①Phase contrast microscope was used to observe the development of cells and the adherence between cells and materials at a fixed time every day. ②Cellular development on days 1, 3, 6 was observed by scanning electron microscope. ③Cellular proliferation was detected by MTT method. ④Alkaline phosphatase (ALP) activity was determined by chemical colorimetry.⑤Flow cytometer test: The effects of PLGA and β-TCP on the cellular cycle, content of DNA and polyploid levels of BMSCs were investigated. The DNA index of the candidate cells was also calculated.RESULTS: ①Phase contrast microscope observation: In the blank control group, cells culture for 7-10 days presented a larger number of shuttle-shaped, and no contact inhibition effect was observed. The time of cells beginning adherence in PLGA group was obviously later than that in β-TCP group. Cells began to develop on the circumambience and surface of the materials, with the prolonging of culture time. Most of the cells were multangular. The cells in both PLGA and β-TCP groups were similar to those in control group in items of cellular development and shape. ②Scanning electron microscope observation: On the seventh day of culture, the cells of control group remained a monolayer and amalgamated to be a patch with multangular-shaped ones increasing. There were substances in granule shape on the surface of the cells and micro-silk links between cells. In PLGA group, the cells After 7 days' cultivation proliferated sharply, and the compressed-shaped ones were inosculated to be a patch through integrations among cells, resulting in a large number of matrixes. While in β-TCP group, the number of cells increased from the 7th day. The cells were combined together to be a monolayer and moved to pores with matrixes creating out of cells.③Cellular proliferation: The number of the cells in each group all increased to some extents. However, there was no significant difference between the PLGA group and the control group, as well as the PLGA group and the β-TCP group (P ＞ 0.05).④ALP activity: The　content of ALP in all the groups enhanced without exception, while the activities between the PLGA group and the Both control group, as well as the PLGA group and the β-TCP group had no significant difference (P ＞ 0.05).⑤Cellular cycle:Both PLGA and β-TCP had slight effects on cellular cycle of BMSCs. The cells in each group were all normal diploid,and no heteroploid cells were discovered.CONCLUSION: This type of PLGA scaffold possesses good cellular biocompatibility, and can be used as a carrier of many factors in bone tissue engineering.

Synthesis of a bio-active bone-matrix material and study of the cellular biocompatibility / 生物医学工程学杂志

To prepare poly(lactic acid/glycolic acid/ asparagic acid-co- polyethylene glycol) (PLGA-[ASP-PEG]) and examine the cellular biocompatibility. PLGA-[ASP-PEG] was obtained by bulk ring-opening copolymerization method, examined by infrared spectrometry (IR) and 1H nuclear magnetic resonance spectroscopy (1H NMR). Bone marrow stromal cells(BMSCs) were cultured with PLGA-[ASP-PEG] (experiment gruop) and PLGA (control group) in vitro respectively, and were observed by phase-contrast microscopy and scanning electron microscopy. The resuls showed that PLGA-[ASP-PEG] was obtained and proved by IR and 1H NMR. The BMSCs of the experiment group could well attach to and extend on the surface of the PLGA-[ASP-PEG], and could proliferate and secrete better extracellular matrix, compared with control. The PLGA-[ASP-PEG] has good cellular a biocompatibility. It can be used as a biomaterial for bone tissue engineering.

Preparation and degradation of poly(DL-lactide)/calcium phosphates porous scaffolds / 生物医学工程学杂志

The porous foams were prepared by the solvent-casting and particulate-leaching technique using poly(DL-lactide) (PDLLA), poly(DL-lactide)/hydroxyapatite (PDLLA/20wt%HA), and poly(DL-lactide)/beta-tricalcium phosphate(PDLLA/20wt% beta-TCP) respectively. Observations by scanning electron microscopy indicated that the HA and beta-TCP were homogeneously dispersed in the polymer matrix, and the pores of the foams are interconnected, resulting in continuous pore structures. The porosity of PDLLA/HA and PDLLA/beta-TCP foams was lower than that of the pure PDLLA foams, but the compression strength was higher than that of the pure PDLLA foams. The results of the degradation in vitro showed that both HA and beta-TCP had significant inhibitory effects on the degradation of PDLLA, especially the HA. It is expected that the composite foams are of use as scaffolds for bone tissue engineering.

The dose-effect correlation between tissue engineered bone loaded BMP at different densities and new bone formation in vivo / 中华骨科杂志

Objective To investigate the effect of a kind of tissue engineered bone implants on healing of segmental bone defects. Methods A 15 mm bone defect experimental model at the right radius of New Zealand white rabbit was made. 24 animals of this model were divided into three groups at random. PLGA scaffords loaded with 5 mg BMP and about 1?106 BMSCs were implanted in Group 1(n=10); PLGA scaffords loaded with 2.5 mg BMP and about 1?106 BMSCs were implanted in Group 2(n=7); PLGA scaffords loaded with 1 mg BMP and about 1?106 BMSCs were implanted in Group 3(n=7). The osteogenesis at the defect area was observed by regular roentgenography and X-ray analysis, histological changes at the bone defects at 4th, 8th, 12th weeks after operation were studied and the new bone formation was measured by image analysis. Biomechanical analysis was done 12 weeks after operation. Results A large quantity of callus was found in all the 3 groups 4 weeks after repair. At the 12th week, the complete bone healing rate was 7/8, 3/5, 3/5, respectively in Group 1, 2 and 3. Group 1 had the largest quantity of new bone measured by histopathological study and X-ray analysis, and at the 12th week, the medullary cavity in this group became united. The compress stiffness, torsion stiffness and the maximal load in Group 1 were better than those in other groups. Conclusion The tissue engineered bone contructed by PLGA scaffords that are loaded with 5 mg BMP and BMSCs is capable of repairing segmental bone defects.