OBJECTIVE To investigate the methods to fabricate repair materials of tissue engineered peripheral nerve with bioactivity of Schwann cells (SC). METHODS 1. The materials were made by dry-wet spinning process to fabricate PLA hollow fiber canal with external diameter of 2.3 mm, internal diameter of 1.9 mm, thickness of 0.4 mm, pore size of 20 to 40 microns, pore ratio of 70% and non-spinning fiber net with pore size of 100 to 200 microns, pore ratio of 85%. 2. SC were implanted into excellular matrix (ECM) gel to observe the growth of SC. 3. SC/ECM complex were implanted into non-spinning PLA fiber net to observe the growth of SC. 4. SC, SC/ECM and SC/ECM/PLA were implanted into PLA hollow fiber canal to bridge 10 mm defect of rat sciatic nerve. RESULTS 1. SC were recovered bipolar shape at 1 day after implantation, and could be survived 14 days in ECM gel. 2. After SC/ECM complex was implanted into PLA net, most of SC were retained in the pore of PLA net with the formation of ECM gel. SC could be adhered and grown on PLA fiber. 3. Most of SC in ECM gel could be survived to 21 days after transplantation. Survival cell numbers of SC/ECM and SC/ECM/PLA groups were obviously higher than SC suspension group. CONCLUSION Non-spinning PLA porous biodegradable materials with ECM is benefit for SC to be adhered and grown.
Objective To review the research advances of the seed cells , the scaffolds and the tissue fabrication of tissue engineering bone. Methods The literature concerned culturing human bone marrow cells, reshaping cuttlebones, and fabricating bone tissues was reviewed extensively. Results Osteoblasts from human bone marrow were successfully induced and differentiated; the cuttlebones were reshaped in cuttlebone hydroxyapatite for the first time; and some tissue engineering bones, such as the osteoblasts/CHA500R, osteoblasts/cuttlebone hydroxyapatite and osteoblasts/PBH were fabricated. Conclusion These tissue einineering bones construced by marrow-derived osteoblasts and CHA500R, cuttlebone hydroxyapatite or PBH are hopeful to be applied clinically.
OBJECTIVE: To explore the relationship between characteristics of transformed cell and tumorigenicity. METHODS: Documents about transformed cell and tumorigenicity were reviewed in detail. RESULTS: Normal biological characteristics and cell function could be maintained in non-tumorigenic transformed cell, but it was changed markedly in malignant transformed cell. CONCLUSION: Non-tumorigenic transformed cell can be served as a standard cell line to study the function and growth characteristics of normal cell.
ObjectiveTo comprehensively analyze the recent advancements in the field of mesenchymal stem cells (MSCs) aging,and summary its achievements and its difficulty at the present. MethodsThe literature about MSCs aging was reviewed and analyzed. ResultsInducible telomerase reactivation of MSCs is successful to extend the life span of senescent cells,but it also has potential safety hazard.The age range presented in the research of age-related cell senescence is inconsistent,resulting in different outcomes.Many ways to improve cell in vitro culture conditions will help delay aging.Recent research indicates that oxidative stress theory is seemed to not completely explain cell aging. ConclusionFurther research of MSCs aging mechanism will help the tissue engineering transform to clinical application.
Objective To fabricate a novel porous bioactivecomposite biomaterial consisting of poly lactic acid (PLA)bone matrix gelatin(BMG) by using the supercritical carbon dioxide fluid technique (SC-CO2) and to evaluate its osteoinductive activity. Methods The cortical bones selected from healthy adult donors were processed into BMG by the defatting, demineralizing, and deproteinizing processes. PLA and BMG were mixed at a volume radio of 3∶1; then, the PLA-BMG mixed material and the pure PLA material were respectively placed in the supercritical carbon dioxide reaction kettles, and were respectively added by the NaCl particles 100200 μm in diameter for theporosity of the materials so that the porous PLA-BMG composite material and the porous PLA composite material could be formed. The mouse osteoblastlike MC3T3-E1 cells were cultured in the dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum. Then, 20 μl of the MC3T3E1 cell suspensions containing 2 ×106 cells /ml were delivered into the culturing plate (24 wells/plate) made of the different materials, which were co-cultured for 2 weeks. In the PLA-BMG group, 100 μg of the crushed PLA-BMG material was contained in each well; in the PLA group, 100 μg of the crushed PLA material was containedin each well; and in the DMEM group, only DMEM was contained, which served as the control group. There were 6 wells in each group. The quantitative analysis onthe calcification area was performed by the staining of the alizarin red S. Theco-cultured cells were harvested and lysated in 1 ml of 0.2% Nonidet P-40 by the ultrasonic lysating technique. Then, the ALP activity and the Ca content were measured according to the illuminations of the reagent kits. Results The porous PLABMG composite material showed a good homological porosity with a pore diameter of 50-150 μm and a good connectivity between the pores. The ALP activity, the Ca content, and the calcification area were significantly greater in the PLABMG group than in the PLA group and the control group (325.59±70.40 U/gprot, 3.51±1.64 mmol/gprot, 42.98±4.44% vs. 63.62±30.01 U/gprot, 1.04±0.21 mmol/gprot, 9.55±1.94%, and 2.40±1.47 U/gprot, 0.70±0.24 mmol/gprot, 0.86±0.41%; Plt;0.05). Meanwhile, there was a statistically significant difference between the PLA group and the control group in the ALP activity and the calcification area (Plt;0.05). Conclusion The porous PLABMG composite material prepared by the use of SC-CO2 has a good steoinductive activity and can be used as a promising bone biomaterial and a bone tissue engineered scaffold.
Objective To evaluate the biocompatibility of a new bone matrix material (NBM) composed of both organic and inorganic materials for bone tissue engineering. Methods Osteoblasts combined with NBM in vitro were cultured. The morphological characteristics was observed; cell proliferation, protein content and basic alkaline phosphatase(ALP) activity were measured. NBM combined with osteoblasts were implanted into the skeletal muscles of rabbits and the osteogenic potential of NBM was evaluated through contraat microscope, scanning electromicroscope and histological examination. In vitro osteoblasts could attach and proliferate well in the NBM, secreting lots of extracellular matrix; NBM did not cause the inhibition of proliferation and ALP activity of osteoblasts. While in vivo experiment of the NBM with osteoblasts showed that a large number of lymphacytes and phagocytes invading into the inner of the material in the rabbit skeletalmuscle were seen after 4 weeks of implantation and that no new bone formation was observed after 8 weeks. Conclusion This biocompat ibility difference between in vitro and in vivo may be due to the immunogenity of NBM which causes cellular immuno reaction so as to destroy the osteogenic environment. The immunoreaction between the host and the organic-inorganic composite materials in tissue engineering should be paid more attention to.
Objective To investigate effects of the basic fibroblast growth factor (bFGF) and fibronection (FN) on the osteoblast adhesion on the bio-derived bone. Methods The third generation of the osteoblast was treated with bFGF 0.1, 1, 10, and 100 ng/ml, respectively, and then was seeded in the bioderived bone, which had been modified with FN 0.1, 1, 10, and 100 μg/ml, or Polylysine, respectively. The cell adhesion was measured by the MTT assay. The cell density and the cell appearance were observed by the scanning electron microscope. The abovementioned procedures were repeated by an application of the GRGDS peptide. Results Both FN and bFGF could enhance the osteoblast adhesion efficiency on the bioderived bone (Plt;0.05). However, the osteoblast adhesion efficiency could be significantly strengthened bya combined use of FN and bFGF. FN and bFGF had a significant synergistic effectin statistics (Plt;0.01), but Polylysine and bFGF had no such synergistic effect (P＞0.05). The combined use of FN and bFGF had a better effect on the cell density and the cell appearance than either of them when observed with the scanning electron microscope. Adhesion efficiency generated by the combined use of FN and bFGF was significantly blocked by the application of the GRGDS peptide. Conclusion The combined use of FN and bFGF has a significant synergistic effect on the osteoblast adhesion efficiency on the bioderived bone. This effect is probably mediated by the RGD-integrin α5β1 pathway.
Objective To evaluate the feasibility of poly-L-lactide(PLLA)/porcinederived xenogeneic bone(PDXB) composite as a scaffold for the bone tissue engineering. Methods The film and the scaffold of the PLLA-PDXB composite were respectively prepared by a solution casting method and a solution casting-particle leaching method. The composite film and scaffold were further treated by the surface alkaline hydrolysis. The surface morphology of the composite was observed by the scanning electron microscopy, and hydrophilicity degree of the composite was measured. The OCT-1 osteoblastlike cells were cultured and amplified in vitro as the seeding cells, which werethen implanted on the film and scaffold. The adherence rate, adherence shape,proliferating activity, and growing morphology of the OCT-1 osteoblastlikecells were observed on the film. Results The PDXB particle 50 μm in diameter on average had a similar phase structure to that of hydroxyapatite. But its Ca/P ratio was lower than that of hydroxyapatite. After the surface alkaline hydrolysis, the PDXB particle could be exposed on the surface of the PLLA-PDXB composite. The surface roughness and hydrophilicity of the PLLAPDXB composite were obviously enhanced. The cell adherence rate and the cell proliferation activity of the PLLAPDXB composite were higher than those of the pure PLLA material. The cells tended to grow on the exposed surface of the PDXB particles. The cells seeded on the composite scaffold could migrate to the inside of the composite scaffold and grew well. Conclusion The PLLA-PDXB composite has a good cell affinity, and this kind of composite can hopefullybecome a new scaffold material to be used in the bone tissue engineering.
Objective To build artificial dermis by using the acellular dermis matrix(ADM), collagen membrane and collagen gel as scaffolds. Methods The fibroblasts were isolated by enzyme from infant skin and were cultivated in the DMEM medium. After 14 days when the fibroblasts were seeded into 3 different scaffolds, the autografts were detected by HE staining, transmission electron microscope and scanning electron microscope. Results ①The fibroblasts obtained from the fullskin by enzyme could be passaged in the Dulbecco’s modified Eagle’s medium 2high gluco se w ith 10% calf bovine serum. ②A layer of fibroblastsw ere found on the surface of th ree different scaffo lds, the fibroblasts could grow into the co llagen membrane and the co llagengel, but could no t be found in the inner of ADM. ③A rt ificial derm is cont racted slightly by inoculat ing fabricat ion on collagen membrane and ADM , and the fibroblasts on them w ere no t act ive in proliferat ing; but the art ificial derm is built by the collagen gel cont racted obviously. Conclus ion The art ificial dermis built by ADM , collagen membrane and collagen gel as scaffolds have a preferable structure for an ideal subst itute of sk n, and can beused as the graft in the next experiments.
Objective To explore a method to isolate, culture and multiplicate the placentaderived mesenchymal stem cells (PMSCs) and the bone marrow-derived mesenchymal stem cells (BMSCs) of rabbit，and to compare their biological characteristics. Methods PMSCs were isolated from placenta of 1fetation rabbitby Percoll density gradient centrifuge and cultured in vitro. BMSCs were isolated from hindlimb bone marrow blood of 1 new born rabbit by direct plates culturemethod. The 3rd passage PMSCs and BMSCs were observed by inverted phase contrast microscope. The stem cell marker (CD44, CD105, CD34 and CD40L) were examined by immunohistochemistry. The 2nd passage PMSCs and BMSCs were co-cultured with biomaterials，(1.0-1.5)×106 cells in one biomaterial， and then observed by aematoxylinstaining after 5 days,and by SEM after 3 days and 8 days. Results PMSCs and BMSCs were both uniformly spondle-shaped in appearance and showed active proliferative capacity. The proliferative ability of PMSCs were quite b and declined with passages. After cultured 10 passages in vitro, its growthslowed. Both PMSCs and BMSCs expressed CD44 and CD105,but did not express CD34 and CD40L immunoreactivity. PMSCs and BMSCs poliferated and adhered to the surface of biomaterials, and cell formed clumps and network; the cells proliferation and the matrix were seen in the pore after 5 days of culture. The observation ofSEM showed that many cells adhered to the biomaterials with spindle-shape and polygon after 3 days; and that PMSCs and BMSCs grew,arranged in layers andsecreted many matrices; the reticular collagen formed arround cells after 8 days. Conclusion PMSCs and BMSCs have similar biological characteristics and PMSCs can be served as excellent seedingcells for tissue engineering.