Effects of strontium-doped calcium polyphosphate on behavior and angiogenic growth factors expression of co-cultured osteoblasts and endothelial cells / 中国组织工程研究

BACKGROUND:Our previous studies have shown that strontium-doped calcium polyphosphate containing low-dose strontium appears to have a significant effect on angiogenesis-related behaviors of monocultured umbilical vein endothelial cells and osteoblasts. OBJECTIVE:To investigate the effect of strontium-doped calcium polyphosphate on angiogenesis-related behaviors of umbilical vein endothelial cells and osteoblasts co-cultured, including celladhesion, spreading, proliferation, as wel as the protein secretion of vascular endothelial growth factor and basic fibroblast growth factor from co-culture system in vitro. METHODS:Human umbilical vein endothelial cells and osteoblastic cells (MG63) were utilized in this study. cells from passage 3 were used for preparation of the cel-scaffold constructs. After placed in 24-wel plate at a ratio of 2:1, human umbilical vein endothelial cells and MG63 cells were seeded onto strontium-doped calcium polyphosphate, calcium polyphosphate and hydroxyapatite scaffolds and co-cultured for 7 days. The vascular endothelial growth factor and basic fibroblast growth factor protein levels were determined through a double ligand enzyme-linked immunosorbent assay. The colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay was performed to quantify the effect of scaffolds on cellproliferation. RESULTS AND CONCLUSION:Compared with those on calcium polyphosphate and hydroxyapatite scaffolds, cells on strontium-doped calcium polyphosphate scaffolds attached and spread better with a significantly improved cellproliferation. More importantly, the vascular endothelial growth factor and basic fibroblast growth factor expressions were significantly higher in the strontium-doped calcium polyphosphate group than the other two groups (P<0.05), indicating strontium-doped calcium polyphosphate can up-regulate levels of vascular endothelial growth factor and basic fibroblast growth factor proteins.

Preparation and properties of porous co-substituted calcium polyphosphate scaffold as bone repair material* / 中国组织工程研究

BACKGROUND: Ions doping is an important method for the modification of bioceramic.OBJECTIVE: To evaluate a novel co-substituted bioceramic scaffolds as bone repair material.METHODS: The microstructure and crystallization of the scaffolds were detected by scanning electron microscope and X-ray diffraction. Compression strength test,degradation test and cell culture experiment were assumed to evaluate the properties of KSCPP in vitro. After a short period of muscle pouches implantation,the performance of KSCPP in vivo was evaluated.RESULTS AND CONCLUSION: The results show that KSCPP scaffold has a higher compressive strength and degradation rate. Moreover,the MTT assay and implantation test reveal that the KSCPP scaffold exhibits lower cytotoxicity and better tissue biocompatibility than CPP and HA. The study proved the great potential of KSCPP in bone repair applications.

Study on preparation of acellular matrix material fixed by oxidized sodium alginate and its cytocompatibility / 生物医学工程学杂志

This study was intended to investigate the crosslinking characteristics of a new crosslinking agent-oxidized sodium alginate (ADA), which might provide an ideal biological crosslinking reagent for the construction of soft tissue bioprostheses. Glutaraldehyde and genipin, which have been typically used in developing bioprostheses, were used as controls. The porcine aortas were treated by these three crosslinking agents for 15 min to 72 h and the fixation index was determined. Subsequently, the mechanical property and cytocompatibility of fixed tissues were also tested. The results indicated that fixed tissues by ADA were comparable as glutaraldehyde and superior to genipin controls in fixative efficiency. It was also found that tissues fixed by ADA were comparable as genipin and superior to glutaraldehyde controls in cytocompatibility and were similar to natural tissues in mechanical property. The results of in vitro study demonstrated that ADA could be a promising crosslinking reagent for biological tissue fixation.

Preparation and properties of calcium polyphosphate-based composite scaffold for bone tissue engineering / 生物医学工程学杂志

Calcium polyphosphate (CPP) is a new type of degradable material for bone repair, yet it is fragile and is not so controllable in regard to degradation. For increasing biological activity and close proximity to natural bone structure, in this experiment, we chose chitosan (CS) and its derivative carboxymethyl chitosan (CMC) as the extracellular matrix structure for the organic phase. Aldehyde sodium alginate (ADA) was used as natural cross-linker. The binary (CPP/CMC) and ternary (CPP/CMC/CS) composite scaffolds were prepared by the "multiple composite-cross-linking method". The degradation laws of the two materials were investigated through the weight loss of scaffolds, the pH value of degradation solution, the compressive strength and the surface morphology characterization. The results showed that the composite scaffolds had good interface and the compressive strength increased greatly, but the organic phase of dual-phase composite scaffolds degraded quickly, while degradation controllability and mechanical properties of ternary composite scaffold were significantly improved. All the above findings show that the method of ternary complex scaffold preparation is useful for the design and preparation of bone tissue engineering materials.

Effect of strontium-doped calcium polyphosphate on endothelial cells-derived angiogenic factor matrix metalloproteinase-2 expression / 中国组织工程研究

BACKGROUND: Strontium-doped calcium polyphosphate (SCPP), as a new repairing material, has been demonstrated to have favorable biocompatibility and biodegradability and some effects on promoting self-angiogenesis. However, the mechanism remains still unknown. OBJECTIVE: Endothelial cells were cultured with SCPP scaffolds in vitro, as well as the cell proliferation and angiogenic factor matrix metalloproteinase-2 (MMP-2) secretion were observed. DESIGN,TIME AND SETTING: A contrast study was performed at the Laboratory of Tissue Engineering of Sichuan University from September 2008 to April 2009. MATERIALS: A series of calcium polyphosphate (CPP) respectively containing 1 %, 2%, 5%, 8%, and 10% Sr~(2+) were prepared.METHODS: ① Materials were plated on 24-well culture plate,and endothelial cell suspension (300 μL) were seeded on 24-well culture plate at the concentration of 3×10~7/L and cultured with 200 uL RPMI1640 culture media. Endothelial cell proliferation was observed using MTT method at days 1,3,5, and 7 after culture. ② CPP and 8% SCPP were plated on 24-well culture plate, and endothelial cell suspension (300 uL) was then incubated in 24-well culture plate at the concentration of 1x10~8/L and cultured with 600 uL RPMI1640 culture media. The morphology of endothelial cells was observed by scanning electron microscopy (SEM) at day 5 after culture.③ Endothelial cells were co-cultured with SCPP of various Sr~(2+) contents for 5 days. After confluence, cells were centrifuged to obtain supernatant. Angiogenic factor MMP-2 secretion was evaluated by ELISA assay.MAIN OUTCOME MEASURES: The proliferation and morphology of endothelial cells on SCPP and CPP were observed. The amount of endothelial cells-derived MMP-2 protein secretion was detected. RESULTS: MTT method demonstrated that the proliferation of endothelial cells on the 8% SCPP scaffold showed a higher level compared to CPP, and other SCPP groups. Scanning electron microscope results suggested that endothelial cells on 8% SCPP had a better growth status and biological activity. ELISA results indicated that angiogenic factor MMP-2 expression on the SCPP was promoted compared with that of CPP, and 8% SCPP showed the highest expression (P < 0.05). CONCLUSION: SCPP has good compatibility with endothelial cells,promoting angiogenesis and enhancing the angiogenic factor MMP-2 expression.

Effect of strontium-doped calcium polyphosphate on the secretion of vascular endothelial growth factor from osteoblastic cells / 中国组织工程研究

BACKGROUND: Strontium-doped calcium polyphosphate (SCPP) is a new type of bone repair materials with good biocompatibility and controlled degradation. The preliminary studies of our group indicate their role in promoting angiogenesis,but its mechanism is unclear.OBJECTIVE: By co-culturing osteoblasts ROS17/2.8 with SCPP in vitro to observe cell proliferation and the secretion of vascular endothelial growth factor (VEGF).DESIGN, TIME AND SETTING: A contrast study was performed at the Laboratory of Tissue Engineering of Sichuan University from October 2008 to June 2009.MATERIALS: A series of calcium polyphosphate (CPP) respectively containing 0%, 1 %, 2%, 5%, 8%, and 10% Sr~(2+) were prepared. ROS17/2.8 osteoblastic cell strain was provided by Laboratory of Transplantation Immunity and Transplantation Engineering, West China Hospital, Sichuan University.METHODS: ①Preparation of cell scaffold complexes: The materials were placed in 24-well plates, then 300 μL cell suspension with a concentration of 2×10~7 cells/Lwas inoculated into each hole. These complexes were cultured for 14 days and the liquid was changed every two days. ②These complexes were measured by MTT assay to observe the proliferation of osteoblasts on the 1~(st), 3~(rd), 5~(th), 7~(th), 10~(th) and 14~(th) days, respectively. ③ The centrifugal supernatant of the complex cultured for seven days was measured by ELISA assay to check the secretion of VEGF.MAIN OUTCOME MEASURES: The proliferation of osteoblastic cells on SCPP and CPP was observed. The amount of VEGF protein secreting from osteoblastic cells was detected.RESULTS: The results of MTT showed that, compared with the CPP group, SCPP groups could promote the proliferation of osteoblasts, and 8% SCPP group was the best; ELISA results showed that, compared with the CPP group, SCPP groups could increase the amount of VEGF protein secretion, of which the promoting role of 8% SCPP was the most obvious (P < 0.05).CONCLUSION: When cultured with osteoblasts, SCPP can promote cell proliferation, and can significantly increase the secretion of VEGF; moreover, 8% SCPP is the best, which reveals a certain mechanism of its promoting angiogenesis.

Effect of processing parameters on the degradation of calcium polyphosphate bioceramic for bone tissue scaffolds / 生物医学工程学杂志

This study was undertaken to elucidate the degradation regularity of calcium polyphosphate (CPP) scaffolds with different preparation parameters. CPP scaffolds with different main crystalline phases were prepared by controlling the particle size of the calcining stuff and the calcining heat. Specimens were soaked into Tris-buffer solution and simulated body fluid (SBF) for 60 days. Results show: alpha-CPP degrades faster than does beta-CPP, and beta-CPP degrades faster than does gamma-CPP; the lower the sinter temperature, the better the degradation of CPP morever, the degradation rate of CPP is inversely proportional to the original particle size. These data suggest that crystal type, sinter temperature and particle size influence the degradation rate of CPP markedly.

Structure and performance of calcium polyphosphate for bone tissue engineering / 生物医学工程学杂志

Porous calcium polyphosphate (CPP) has shown promise of tissue engineered implant application because of the biocompatibility and biodegradation. CPP with different polymerization degree were prepared by controlling the calcining time, and its polymerization degree could be calculated by developed method in this paper. Different crystal types CPP were prepared by quenching from the melt and crystallization of amorphous CPP. From the in vitro degradation, carried out in Tris-HCl buffer, the degradation velocity of CPP was controllable. The weight loss of CPP with different polymerization degrees and crystal types were different. With the increasing of polymerization degree, the weight loss during the degradation was decreasing, contrarily the strength of CPP was increasing. The amorphous CPP could degrade completely in 17 days while gamma-CPP do completely in 25 days. The degradation velocity beta-CPP and alpha-CPP was slower than gamma-CPP and the weight loss was about 12% and 5% respectively. The results of this study indicate that CPP have potential applications for bone tissue engineering as inorganic polymeric biomaterials.

Methods for the pre-treatment of biological tissues for vascular scaffold / 生物医学工程学杂志

Today, the blood vessel substitutes are in large demand for coronary and peripheral bypass procedures, and the demand cannot be met by conventional sources. This problem will be solved by applying tissue-engineered blood vessel in clinics. The prefabrication of vascular scaffold will be involved in engineering a blood vessel substitute. Biological tissues are important biomaterials fabricating vascular scaffold which can offer better constructs for adhesion and growth of cells onto synthetic materials. Because of immediate degradation of biological tissues obtained from the abattoir, cadaver or patient and the presence of antigenicity in allogenic or xenogenic tissues, the fresh biological tissues can not directly be preserved and applied. The use and preservation of these natural biomaterials have typically required pre-treatment aimed at (1) reducing the antigenicity of the materials, (2) enhancing the resistance of the materials to enzymatic degradation, (3) stabilizing the structure of the tissues and maintaining their mechanical properties. Physical and chemical methods for the pre-treatment of biological tissues are available. The predominant chemical agents that have been investigated for the pre-treatment of biological tissues for vascular scaffold are glutaraldehyde, polyepoxy compound, carbodiimide, genipin and proanthocyanidin. Typical and particularly promising physical pre-treatment of biological tissues for vascular scaffold is dye-mediated photooxidation. The crosslinking mechanisms of all classes of pre-treatments and the effects of pre-treatments on antigenicity, biostability, mechanical properties, cytoxicity and calcification of treated tissues are described in this article. The advantages and disadvantages of all pre-treatments are also reviewed. The trend of pre-treatment of biological tissues is to investigate and exploit the naturally occurring crosslinking reagent with less cytoxicity. Meanwhile, dye-mediated photooxidation crosslink is also a promising pre-treatment which should be widely applied in vascular scaffold.

Synthesis and application of the polyacrylamide beads acting as LDL adsorbent's matrices / 生物医学工程学杂志

This study in pursuit of the synthetic technologies and structure characterization of polyacrylamide-based matrices (PAM beads) for low density lipoprotein (LDL) adsorbent and their adsorbability for LDL was intended for an experimental evidence of developing advanced matrices for LDL adsorbent. PAM beads were synthesized by inverse suspension polymerization, and their structure characterization was characterized by SEM, image analyzer and small angle X-ray scattering. The tripeptide serine-aspartic-glutamic acid (SDE) was coupled on the PAM beads to prepare the LDL adsorbents whose adsorbability for LDL was determined in vitro. The results showed that the PAM beads with the average size diameter 142.1 microm and the average pore diameter 119.8 nm could act as the matrices in accordance with the requirement of adsorbent for LDL. When the amount of acrylamide and the crosslinking agent N,N'-methylene-bis(acrylamide) was fixed, the average pore diameter decreased with the increase of the crosslinking agent content. Although the nonspecific binding of PAM beads for LDL was low, they could selectively adsorb LDL after coupling the SDE on the PAM beads.

Peptides used in lowering the level of LDL in plasma when treating familial hypercholesterolemia / 生物医学工程学杂志

While introducing the indications of low-density lipoprotein (LDL) apheresis, LDL absorption systems were reviewed generally. As the key components for binding LDL, four kinds of ligands which are synthesized by different principles are: 1. Positively charged peptides designed according to state charge force between ligand and LDL; 2. Peptides designed according to structural characteristics of the binding site between LDL and its receptors; 3. Antibody of Lp (a) obtained by immunizing mammals with designed peptides with the characteristics of Lp (a); 4. Segments of LDL binding proteins (LBPs) synthesized with genetic engineering method based on the specific binding of LBPs to LDL. Requirements of matrices carrying these ligands are also considered. Finally, future developments in treatments of familial hypercholesterolemia by means of blood purification using synthesized peptides are overlooked.

Experimental Study of Preparation and Characteristics of Biomaterials Fabricating Esophageal Prosthesis / 医疗卫生装备

Objective To investigate the characteristics and cytocomptibility of porcine aorta fixed with genipin in vitro and to provide the proper biomaterials for fabrication of esophageal prosthesis. Methods The porcine aortas were treated by genipin for 7min to 7days, were then examined with naked-eye and light microscope. Subsequently, the fixation index determination and the biomechanics test were also performed. The cytotoxicity of the genipin-fixed materials was evaluated in vitro by MTT assay using a mouse -derived established cell line of L-929 fibroblasts as test cells. Results The experimental results indicated that the antigenicity of porcine aorta tissues could be diminished by genipin through getting rid of cell in the porcine aorta tissues and reducing the level of free amino groups in the porcine aorta tissues. The structural integrity of porcine aorta tissues can be preserved after treating with genipin. It was also found that the genipin-fixed porcine aorta tissues appeared more toughness and visco-elasticity. The RGR of genipin-fixed porcine aorta tissues was high which presumed a low cytotoxicity of this material. Conclusion The Genipin-fixed porcine aorta should be a promising material for fabricating esophageal prosthesis or fabricating scaffold of tissue-engineered esophagus.