Cellular immune response and immune toxicity to BALB/c mice for animal-based collagen / 中国组织工程研究

BACKGROUND:Natural colagen is considered to have low immunogenicity and good biocompatibility relatively. OBJECTIVE:To evaluate the immunogenicity of animal-based colagenin vitro. METHODS:Type I colagen was extracted from bovine tendon after immunogenicity removal. The colagen purity was detected by high performance liquid chromatography and residual DNA was measured quantitatively by fluorescence staining. Fifty BALB/c mice were randomly divided into five groups: subcutaneous injection of normal saline solution (negative control), bovine colagen (positive control), 33.4, 66.8, 133.4 mg/kg of bovine tendon colagen, respectively, once a day. After 12 days of continuously subcutaneous injection, lymphocyte proliferation, and cel classification and NK cel kiling function of mice were detected; after 3 weeks of continuous injection, the spleen, liver, spleen and lung tissue of mice were taken for histological examination. RESULTS AND CONCLUSION:Compared with the standard type I colagen, the purity of purified type I bovine colagen reached more than 99%, but the residual DNA was below 1 mg/L which was far less than the residue level of conventional cel-free DNA in the matrix (dry weight: 50-100 μg/g). After 12 days of continuous injection, there were no changes in lymphocyte proliferation, NK cel kiling function and the proportion of lymphocyte subsets. After 3 weeks of injection, the spleen and lymph sheath of mice around the smal artery became thickened in the 66.8 and 133.6 mg/kg bovine tendon colagen groups, which could cause accidental liver injury and lung injury, but the splenic corpuscle germinal center area had no change. These findings indicate that continuously subcutaneous injection of animal-based colagen can cause the lower lymphocyte immune response to the spleen of BALB/c mice, which may cause accidental liver and lung injuries.

Biological properties, degradation and absorption of collagen spongesin vivo / 中国组织工程研究

BACKGROUND:Colagen sponges are applied for hemostatic use, wound healing, and residual cavity filing, which have great values in clinical application and scientific research. OBJECTIVE:To investigate the biological properties, biocompatibility and biodegradability of colagen spongesin vivo. METHODS: The spatial structure, pore diameter and porosity of colagen sponges were characterized by scanning electron microscopy. Transmission electron microscopy was used to observe the conformation of colagen sponges. The secondary structure and thermal denaturation temperature of colagen sponges were analyzed by circular dichroism spectrum. Colagen sponges were implanted intramuscularly into the spinal cord of New Zealand rabbits to observe the degradation and absorption and histological changesin vivo. RESULTS AND CONCLUSION: Colagen sponges had porous structure with varying pore sizes ranging 40-150 μm, the mean pore size of 100 μm, the thickness wal of 1 μm, and a porosity of approximately 95.8%. Colagen sponges had a typical porous structure and periodic light and dark zones. The solution of colagen sponges had a weak positive band near 220 nm and an intense negative band near 206 nm, which indicated a classic triple helix. And the secondary structure and thermal stability of colagen sponges were similar to that of liquid colagen. Colagen sponges began to degrade at 4 weeks, and remained 20% at 12 weeks. These sponges had been associated with foreign body response and inflammation within 2 weeks after implantation. With wound healing, inflammatory reactions gradualy reduced and disappeared. During the implantation and degradation of sponges, no significant fibrous capsule formed and no tissue necrosis occurred at implantation site, indicating that colagen sponges have good performance in bioactivity, biocompatibility and degradation.

Cytocompatibility of calcium metaphosphate nanoparticles / 中国组织工程研究

BACKGROUND:Calcium metaphosphate has excel ent biocompatibility, degradability, and cel affinity. Human bone marrow mesenchymal stem cel s can grow and proliferate in the pores of the porous calcium metaphosphate, but less is known about calcium metaphosphate nanoparticles. OBJECTIVE:To prepare calcium metaphosphate nanoparticles, and to analyze the effect of calcium metaphosphate nanoparticles at different concentrations on apoptosis of human bone marrow mesenchymal stem cel s by flow cytometry. METHODS:The calcium metaphosphate nanoparticles were prepared by wet bal mil ing. Scanning electron microscopy and transmission electron microscopy were used to observe the morphology of the calcium metaphosphate nanoparticles, and the crystal structure of nanoparticles was analyzed by X-ray diffraction. Calcium metaphosphate nanoparticles were mixed in the CYAGON Oricel TM basal medium, and the concentrations of calcium metaphosphate nanoparticles in the medium were 10, 1, 0.1 mg/L. Human bone marrow mesenchymal stem cel s were cultured for 7 days in the above-mentioned media, and apoptosis of human bone marrow mesenchymal stem cel s was analyzed by flow cytometry. RESULTS AND CONCLUSION:Calcium metaphosphate nanoparticles were successful y prepared by wet bal mil ing, irregular in shape, and the mean diameter was 10-30 nm. X-ray diffraction results showed the crystal structure of nonaparticles was mainlyβ-Ca(PO3)2. The cel ratio of G0/G1 phase and G2/M phase in 10 mg/L group was obviously higher than that in 1, 0.1 mg/L groups (P<0.01). The cel apoptosis rates during the early, middle, late stages in 10 mg/L group were obviously higher than those in 1, 0.1 mg/L groups (P<0.01), and the total cel apoptosis was also significantly increased in 10 mg/L group (P<0.01). These findings indicate that human bone marrow mesenchymal stem cel s proliferation can be inhibited by calcium metaphosphate nanoparticles, and apoptosis rate is increased significantly when the concentration of calcium metaphosphate nanoparticles increases from 1 mg/L to 10 mg/L.

Preparation and cytocompatibility of novel porous calcium metaphosphate composite membranes / 中国组织工程研究

BACKGROUND:Porous poly(3-hydroxybutyrate-co-4-hydroxybutyrate)/calcium metaphosphate composite membranes prepared previously is too thick and uneven in holes. OBJECTIVE:To prepare the thin even porous poly(3-hydroxybutyrate-co-4-hydroxybutyrate)/calcium metaphosphate composite membrane, and to evaluate the cytocompatibility and differentiation capacity. METHODS:Porous and nonporous, thin and even poly(3-hydroxybutyrate-co-4-hydroxybutyrate)/calcium metaphosphate composite membranes were prepared by phase separation method. Its thickness and weight loss rate were determined. Human bone marrow mesenchymal stem cel s were cocultured with porous and nonporous poly(3-hydroxybutyrate-co-4-hydroxybutyrate)/calcium metaphosphate composite membranes for 7 days. Ultrastructure of composite membranes was observed under the scanning electron microscopy. Surface markers of the bone marrow mesenchymal stem cel s on the composite membranes were analyzed using flow cytometry. RESULTS AND CONCLUSION:The thickness of the porous and nonporous composite membranes was (0.041 ± 0.005) mm and (0.058±0.004) mm. Weight loss rates of porous and nonporous composite membranes were respectively 19.93%and 7.64%at 24 hours. Calcium metaphosphate particles were evenly distributed in porous and nonporous composite membrane. Cel s spread entirely, showing spindle shape. Calcium metaphosphate particles were evenly distributed in porous composite membrane. Pore in porous composite membranes was also uniformly distributed, and pore size was about 2-8μm. Cel s spread entirely, showing polygonal shape with multiple tentacles. The tentacles of some cel s entered into the scaffold. CD105, CD90, CD44, CD29 and CD73 expression was detected in porous and nonporous composite membranes. There was no significant difference in cel-positive rate. Poly(3-hydroxybutyrate-co-4-hydroxybutyrate)/calcium metaphosphate composite membranes prepared in this study has good biocompatibility and could not promote cel differentiation.

Modification of seaweed polysaccharide-agarose and its application as skin dressing (III)--skin regeneration with agarose grafting hyaluronic acid sponge / 生物医学工程学杂志

In this paper, a kind of skin dressing, agarose- grafting- hyaluronic acid (Ag-g-HA) sponge was applied to test the modified agarose based scaffold for skin regeneration. The bFGF loading agarose-grafting hyaluronan scaffold had homogenous porosities, and the loaded bFGF was bioactive in 2 weeks. The Ag-g-HA sponge was applied into skin of mice, and it was found that the dressing promoted skin regeneration and no infection and leakage in lesion site took place. H&E staining results showed that the repaired skin was similar to autologous skin. These demonstrate that Ag-g-HA sponge has a promise in skin regeneration.

Application of seaweed polysaccharide-agarose dressing to animal skin regeneration / 中国组织工程研究

BACKGROUND: It has vedfied that seaweed polysacchande-agarose modifiers have their medical application value. OBJECTIVE: To perform skin regeneration trials using sprayable activated agarose/gelatin solution, and to explore the possibility of egarose modifier as skin dressing for skin regeneration. METHODS: To prepare 3% sprayable mixture with the dissolved activated agarose and gelatin at a certain ratio, and then filtrated with millopore for sterilization to prepare activated agarose (egarose degradation for 8 hours) and gelatin degradation, and made into different ratios (1: 0, 1: 1, 1: 2, 1: 3). A total of four rabbits were obtained, and four sites were selected on the back of each rabbit, totally 16 experimental sites. The sprayable activated agarose/gelatin mixture (1: 1, 1: 2, 1: 3) was directly sprayed on the four lesion sites. Sprayable activated agarose for two sites and simple gelatin for two sites served as controls. The effects of the wounds sprayed with,dressing were observed at 4 weeks following surgery. RESULTS AND CONCLUSION: At 7 days following surgery, the cover film had broken in mixture of activated agarose and gelatin at 1: 3, and remaining three were intact. No infection or inflammation occurred in wound of four ratios. Following comparison, the wound was rapidly healed in 1: 2 ratio dressing. The additional gelatin showed promoting effects on wound healing significantly. Hematoxylin-eosin staining demonstrated that skin with the dressing was similar to autologous skin, which verified that sprayable activated agarose/gelatin have a premise in skin regeneration.

Anticoagulation properties of sulfated agarose / 中国组织工程研究

BACKGROUND: Polysaccharide sulfate has bean hot focus in recent research.Regarding anticoagulant property of sulfated polysaccharides, recent studies mainly explored degree Of substitution, and there are rare studies concerning relative molecular mass and spatial structure.OBJECTIVE: To study the anticoagulant activity of the sulfated agarose with different relative molecular masses.METHODS: With formamide as dispersing agent,agarose was sulfated with the method of chlor0sulfonic acid-pyridine.The product was graded and purified with dialysis bag.Two kinds of sulfated agarose with different molecular masses were obtained,and their anticoagulant properties were checked by assays of the activated partial thromboplastin time,thrombin time.RESULTS AND CONCLUSION:.Two kinds of sulfated agarose had similar degree of substitution and sulfation position,whereas their molecular weight was different.The bioassay results of coagulation index demonstrated that the anticoagulant activity of agarose improved with the increasing of molecular mass within a certain range.Relative molecular mass had an important effect on the anticoagulant activity of the sulfated agarose.

Chondrogenesis of bone marrow mesenchymal stem cells, chondrocytes and adipose-derived adult stromal cells in three-dimensional scaffolds / 中国组织工程研究

BACKGROUND: The key factor for cartilage repair is an orient differentiation of seed cells in three-dimensional scaffold; however,clear cartilage is haply observed in the three-dimensional scaffold.OBJECTIVE: To investigate the chondrogenesis of bone marrow mesenchymal stem cells (BMSCs), chondrocytes (CCs) and adipose-derived adult stromal cells (ADAS) in three-dimensional scaffolds METHODS: BMSCs, CCs, and ADAS were primary-cultured from goat bone marrow following trypsin digestion and amplified in monolayer. P1 BMSCs, P3 CCs, and P3 ADAS were implanted into collagen/hyaluronic acid scaffold at different concentrations of 10%, 20%, 50%, 80%, and 100%, and the cells were then three-dimensionally cultured in serum-free culture media. Two weeks later, SO staining and immunohistochemical staining were employed to observe the synthesis between of chondroitin sulfate and type Ⅱ collagen.RESULTS AND CONCLUSION: The seeded cells had chondrogenesis in scaffolds under certain conditions. In details, BMSCs in 20% (W/W) hyaturonan/collagen Ⅰ scaffolds could perform a hyaline-cartilaginous phenotype when they were expanded with less than 3 passages and cultured in a chondrogenic medium, and CCs did better than BMSCs in chondrogenesis with high passage number; however, ADAS just showed a minor chondrogenesis even in a pellet culture. BMSCs and CCs were better than ADAS as seeded cells for cartilage tissue engineering and regenerative medicine.