Objective To provide the chosen scaffold materials for experiment and application of tissue engineering and to detect the properties of the collagenbio-derived bone scaffold material loading WO-1. Methods The purebio-derived bone scaffold material, bio-derived bone scaffold material loading collagen, collagen bio-derived bone scaffold material loading WO-1 were made by use of allograftbone, and typeI collagen, and WO-1. The morphological features, constitute components and mechanical properties were examined by scanning electron microscopy,X- rays diffraction and mechanical assay. Results The bio-derived bone scaffold material maintained natural network pore system; the bio-derived bone scaffold material loading collagen maintained natural network pore system, the surface of network pore system was coated by collagen membrane; the collagen bio-derived bone scaffold material loading WO-1 maintained natural network pore system, thesurface of network pore system was coated by collagen membrane. The pore sizes of the 3materials were 90-700 μm, 75-600 μm and 80-600 μm, respectively, and the porosities were 87.96%, 80.47%, 84.2%. There was no significant difference between them(P＞0.05).The collagen bio-derived bone scaffold material loading WO-1 consisted of ［HA,Ca10(OH)2(PO4)6］. There was no significant difference in the mechanical strength of the three scaffold materials. Conclusion The bio-derived bone scaffold material loading WO-1 is as good as bio-derived bone scaffold material and collagen bio-derived bone scaffold material, and it is an effective scaffold material for tissue engineering bone.
Objective To prepare human acellular amniotic membrane(HAAM) and to measure its cytocompatibility and biocompatibility. Methods HAAM were preparedby chemical detergent-enzymatic extraction. Fresh human amnion was crosslinkedwith glutaradehyde, shaken in 0.5% SDS for 24 hours, and then treated with 0.25%trypsin for 4 hours. The production were freeze-drying and sterilized using ethylene oxide. Human fibroblasts were isolated from embryo and expanded in vitro. The fibroblasts were seeded in HAAM. HAAM and specimen were stained with HE and Mallory, and observed grossly, under light microscopy and scanning electron microscopy. The HAAM were implanted in the back of SD rats. Results There wereno residues of cells in the HAAM (HE, Mallory staining). One side of HAAM had reticular and porous structure, the other side had compact fibrous structure.Pore size was from 10 to 80 nm. The HAAM could be seeded with expanded fibroblasts in vitro,and fibroblasts had the potential of spread and proliferation. The SD rat in the implant test had no death, convulsions and other abnormal response. Conclusion The detergent-enzymatic extraction process can remove cellsand solvable components effectively and preserve the tissue matrix well and keep the reticular structure. The HAAM can be used as an ideal scaffold of biological membrane for tissue engineering.
Objective To study the possibil ity of bone marrow mesenchymal stem cells (BMSCs) differentiation into tenocytes (TCs) under strain stimulation by co-culture of BMSCs-small intestinal submucosa (SIS) composites in vitro. Methods BMSCs were isolated by adherent culture from the bone marrow of 1-week-old SD rats. Inducing method of multiple differentiation and flow cytometry were appl ied to identify the cells. The stress-strain curve of SIS was measured with Instron machine. Purified BMSCs (2nd passage, 2.5 × 105 cells/cm2) were seeded on SIS (3 cm × 1 cm at size) and cultured for 2 daysand then continued for another 5 days under strain stimulation (stretching frequency was 0.02 Hz, action time was 15 minutes/ hour and 12 hours/day, strain ampl itude was 5%) as experimental group, while the BMSCs-SIS composites were sustained static culture as control group. TCs were isolated from tail of 1-week-old SD rats. TCs-SIS composites were cultured under non-strained as positive control group. Scanning electron microscope (SEM) was used to examine the morphological changes of BMSCs after strain stimulation. The contents of Scleraxis and Tenomodulin in supernatant were tested by ELISA kit. Results The BMSCs could be induced to differentiate into osteoblasts and l ipocytes, and showed the results of CD34-, CD45-, and CD90+, which were accorded with the biological characteristics of BMSCs. The failure test of SIS showed that the average elastic strain was 39.5%. SEM observation showed that the strain-stimulated BMSCs had the TCs-l ike morphological characteristics. The contents of Scleraxis and Tenomodul in in supernatant of experimental group, control group, and positive control group were (3.56 ± 0.91) μmol/L and (4.27 ± 1.10) μmol/L, (0.23 ± 0.14) μmol/L and (0.16 ± 0.10) μmol/L, and (14.73 ± 2.30) μmol/L and (10.65 ± 1.51) μmol/L, respectively. There were significant differences among 3 groups (P lt; 0.05). Conclusion Appropriate strain stimulation could induce BMSCsdifferentiate into TCs, and the best conditions of strain stimulation need more experiments.
Objective To study the biological characteristic of rabbit bone marrow mesenchymal stem cells (BMSCs) double-labeled by PKH26 and BrdU in vitro, and to construct tissue engineered cardiac patch in vitro. Methods The BMSCs were harvested from 6-month-old New Zealand rabbits and labeled with PKH26 and BrdU. The growth and fluorescent intensitywere observed by inverted phase contrast microscope, fluorescent microscope, flow cytometry, and MTT detection. Thecharacteristics of double-labeled BMSCs differentiating into osteoblasts and adipocytes, respectively, in vitro were identified by alkal ine phosphatase (ALP) staining, Al izarin red staining, Oil red O staining, immunocytochemical technique of collagen type I, and osteocalcin expression. The labeled BMSCs were seeded on the small intestinal submucosa (SIS) and co-cultured for 5-7 days to construct tissue engineered cardiac patch. The patches were tested by inverted phase contrast microscope, fluorescent microscope, scanning electron microscope, and HE staining to observe the cell prol iferation. Results The double-labeled cells grew well and showed red fluorescence. There was no significant difference in the growth characteristic between the labeled and unlabeled cells. There was no significant difference in the expression of stem cell specific surface antigen between before lebel ing and after lebel ing. After osteogenic induction of labeled BMSCs, ALP staining and Al izarin red staining were positive, and the cells expressed collagen type I and osteocalcin. After adipocytes induction, l ipid droplets could be observed in cytoplasm by Oil red O staining. After the co-culture in vitro for 5-7 days, the double-labeled cells grew well, showing a multi-layer cellular structure on the surface of SIS. Conclusion Rabbit BMSCs can be double-labeled with PKH26 and BrdU stably. The labeled cells still have the potential of self-renewal abil ity and multipotent differentiation abil ity; tissue engineered cardiac patch can be constructed by co-culturing labeled BMSCs and SIS in vitro.
Objective To evaluate the biocompatibil ity of manufactured heterogeneous demineral ized bone matrix(DBM) particles and to provide basis for further experimental study and cl inical application. Methods Heterogeneous DBMparticles A (degreased and demineralized) and B (degreased, demineralized and acellular), particle size from 250 to 810 μm, and leaching l iquor were made with a series of physical and chemical methods from pig l imbs cortical bone. The residual calcium and phosphorus contents of bone particles were measured after degreased and demineral ized. The acute toxicity test, skin stimulating test, pyrogeneous test, hemolysis test, cellular toxicity test and muscular embedded test were carried out according standard toxicological method. Results The contents of calcium and phosphorus in cortical bone were (189.09 ± 3.12) mg/g and (124.73 ± 2.87) mg/g, and in demineral ized bone matrix particles were (3.48 ± 0.09) mg/g and (3.46 ± 0.07) mg/ g. The residual calcium content was 1.87%, of phosphorus was 2.69%. The activity of mice was normal in the acute toxicity test. No animal died and no toxicity symptom or adverse effects were shown within 7 days. The mean weight daily increased showed no statistically significant difference (P gt; 0.05) between two groups after 7 days. Skin stimulating reactions were not found in the two experimental groups and negative control group by intradermal stimulation test. The maximal increase of body temperature in two experimental groups were 0.4℃ , which meet the national standard (lt; 0.6 ). The rate of haemolysis to the leaching liquor was 1.14% (A) and 0.93% (B), which was lower than the national standard (lt; 5%). The cell prol iferation rates of two experimental groups when compared with control group showed no statistically significant difference (P gt; 0.05). The toxicity of DBM particlesleaching liquor was graded from 0 to 1, which means the material has no cytotoxicity. All the animals survived well. There was no tissue necrosis, effusion or inflammation at all implantation sites. For the index of HE and Masson staining, there were no effusion around the material and inflammatory cell infiltrate obviously in two experimental groups. Inflammatory cell infiltrate is sl ight in control group 2 weeks postoperatively. The inflammatory cell infiltration was mitigate gradually over time in two experimental groups after 4, 8 and 12 weeks. New bone and collagen fibers formation were observed when the material was degraded and absorpted. Score evaluation of local cellular immune response at different time after operation of two experimental groups showed no statistically significant difference (P gt; 0.05). Conclusion Heterogeneous DBM has no obvious toxicity, skin irritation, pyrogenicity, and no cytotoxicity with a rate of haemolysis lt; 5%, so it has good biocompatibility and partial osteoinductive.
Objective To study the effect of hypoxia on the prol iferation of hBMSCs and human placental decidua basal is-MSCs (hPDB-MSCs), and to provide the theoretical basis for discovering the new seed cells source for tissue engineering. Methods Density gradient centrifugation method was adopted to isolate and culture hBMSCs and hPDB-MSCs,flow cytometry (FCM) was appl ied to detect cell surface marker. After establ ishing the experimental model of CoC12 chemical hypoxia, MTT method was appl ied to evaluate the prol iferation of hBMSCs and hPDB-MSCs at different time points (6, 12, 24, 48, 72, 96 hours) with various CoC12 concentration (0, 50, 75, 100, 125, 150, 175, 200 μmol/L). Results FCM analysis revealed that hPDB-MSCs and hBMSCs expressed CD9, CD29, CD44, CD105, CD106 and human leucocyte antigen ABC (HLA-ABC), but both were absent for CD34, CD40L and HLA-DR. Compared with hBMSCs, hPDB-MSCs expressed stage-specific embryonic antigen 1 (SSEA-1), SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81 better. The prol iferations of hPDB-MSCs and hBMSCs were inhibited within the first 12 hours under hypoxia condition, but promoted after 12 hours of hypoxia. Compared with the control group, the hBMSCs were remarkably prol iferated 24 hours after hypoxia with CoC12 concentration of 150 µmol/L (P lt; 0.05), while hPDB-MSCs were significantly prol iferated 12 hours after hypoxia with CoC12 concentration of 75 µmol/L (P lt; 0.05). Conclusion Compared with hBMSCs, hPDB-MSCs express more specific surface antigens of embryonic stem cells and are more sensitive to the prol iferation effects of chemical hypoxia, indicating it may be a new seed cells source for tissue engineering.
Objective To study the effect of rat osteoblast conditioned culture medium on the BMSCs differentiation of allogeneic rat and to find a new approach to provide seed cells for bone tissue engineering. Methods BMSCs and osteoblasts were harvested from 10 healthy one-week-old SD rats (male and female, weighing 20-30 g) by adherent method and enzyme digestion method respectively. Cell identification was conducted. Osteoblast conditioned culture medium was prepared by mixing supernatant of osteoblasts at passage 1-5 with complete medium (1:1). Then, BMSCs at passage 2 were co-cultured with osteoblast conditioned culture medium (inducement group) and complete medium (control group), respectively. The morphological changes of co-cultured BMSCs were observed by inverted phase contrast microscope, the growth condition of BMSCs was detected by MTT method, the expressions of ALP, Col I and osteocalcin (OCN) in the cocultured BMSCs were tested by immunohistochemistry staining, and the expressions of Col I and OCN mRNA were detected by RT-PCR. Results In the inducement group, BMSCs grew bigger, changing from long fusiform to flat and polygon with protuberance 7 days after co-culture; the presence of cell colony-l ike growth was observed 9 days after co-culture. Cell growth curve demonstrated that the counts of BMSCs was increased with time, there were more cells in the control group than that of the inducement group, and there was a significant difference in cell counts between the control and the inducement group 4-7 days after co-culture (P lt; 0.05). For the inducement group, ALP staining was positive 12 days after co-culture, the calcium nodules were appeared 18 days after co-culture, Col I and OCN were positive 21 days after co-culture, and the expressions of Col I and OCN mRNA were detected by RT-PCR 21 days after co-culture. Conclusion Rat osteoblast conditioned culture medium can significantly induce the differentiation of allogeneic rats’ BMSCs towards osteoblasts.
Objective To explore the effect of tissue engineered cartilage reconstructed by using sodium alginate hydrogel and SIS complex as scaffold material and chondrocyte as seed cell on the repair of full-thickness articular cartilage defects. Methods SIS was prepared by custom-made machine and detergent-enzyme treatment. Full-thickness articularcartilage of loading surface of the humeral head and the femoral condyle obtained from 8 New Zealand white rabbits (2-3weeks old) was used to culture chondrocytes in vitro. Rabbit chondrocytes at passage 4 cultured by conventional multipl ication method were diluted by sodium alginate to (5-7) × 107 cells/mL, and then were coated on SIS to prepare chondrocyte-sodium alginate hydrogel-SIS complex. Forty 6-month-old clean grade New Zealand white rabbits weighing 3.0-3.5 kg were randomized into two groups according to different operative methods (n=20 rabbits per group), and full-thickness cartilage defect model of the unilateral knee joint (right or left) was establ ished in every rabbit. In experimental group, the complex was implanted into the defect layer by layer to construct tissue engineered cartilage, and SIS membrane was coated on the surface to fill the defect completely. While in control group, the cartilage defect was filled by sodium alginate hydrogel and was sutured after being coated with SIS membrane without seeding of chondrocyte. General condition of the rabbits after operation was observed. The rabbits in two groups were killed 1, 3, 5, 7, and 9 months after operation, and underwent gross and histology observation. Results Eight rabbits were excluded due to anesthesia death, wound infection and diarrhea death. Sixteen rabbits per group were included in the experiment, and 3, 3, 3, 3, and 4 rabbits from each group were randomly selected and killed 1, 3, 5, 7, and 9 months after operation, respectively. Gross observation and histology Masson trichrome staining: in the experimental group, SIS on the surface of the implant was fused with the host tissue, and the inferface between them disappeared 1 month after operation; part of the implant was chondrified and the interface between the implant and the host tissue was fused 3 months after operation; the implant turned into fibrocartilage 5 months after operation; fiber arrangement of the cartilage in theimplant was close to that of the host tissue 7 months after operation; cartilage fiber in the implant arranged disorderly andactive cell metabol ism and prol iferation were evident 9 months after operation. While in the control group, no repair of thedefect was observed 9 months after operation. No obvious repair was evident in the defects of the control group within 9months after operation. Histomorphometric evaluation demonstrated that the staining intensity per unit area of the reparative tissue in the defect of the experimental group was significant higher than that of the control group at each time point (P lt; 0.05), the chondrification in the experimental group was increased gradually within 3, 5, and 7 months after operation (P lt; 0.05), and it was decreased 9 months after operation comparing with the value at 7 months after operation (P lt; 0.05). Conclusion Constructed by chondrocyte-sodium alginate hydrogel-SIS in complex with surficial suturing of SIS membrane, the tissue engineered cartilage can in-situ repair cartilage defect, promote the regeneration of cartilage tissue, and is in l ine with physiological repair process of articular cartilage.
【Abstract】 Objective To explore an effective method to cultivate esophageal mucosa epithel ial cells (EMECs)of canine in vitro, and to observe the biological characteristics of EMECs growing on SIS in order to provide an experimental basis for esophagus tissue engineering. Methods Esophageal tissues were obtained from five healthy dogs aged 2 to 5 weeks under sterile conditions. The primary EMECs were cultivated with defined keratinocyte serum free medium (DKSFM) containing 6% FBS. The morphological characteristics and the growth curve of EMECs of the 2nd generation were observed for 1 to 5 days. The expressions of the EMECs marker (cytokeratin 19, CK-19) were examined by immunocytochemistry. The 2nd generation of EMECs was seeded on SIS and observed by HE staining, immunohistochemical staining, and SEM for 4 and 8 days. Results The primary culture of canine EMECs arranged l ike slabstone. Immunohistochemical staining of CK-19 of the2nd generation EMECs showed positive broadly. The cells growth reached the peak level at 2 days by MTT method. E MECs werepolygon in shape and arranged l ike slabstone, and formed a single layer on the surface of SIS. The cells were contact ed closely with each other for 4 days. Eight days later, 2 to 3 layers stratified structure was formed. Lots of EMECs were grown on SIS, andshowed laminate arrangement. Conclusion With mixed enzymatic digestion, the culture of EMECs in DKSFM containing 6 %FBS is a simple and feasible method. SIS shows good biocompatibil ity and can be used as a good scaffold material in th e tissue engineered esophagus.
Objective To isolate and culture the chondroid cells and notochord cells from New Zealand rabbit immature nucleus pulposus (NP) in monolayer, and to valuate the responsiveness of rabbit disc-derived chondroid cells to notochord cells with respect to cell prol iferation and phenotype. Methods The NP cells were released from the minced immature NP of 6 New Zealand rabbits (4-week-old) by 0.2% collagenase II digestion. The chondroid cells and notochord cells were purified by discontinuous gradient density centrifugation. The chondroid cells were cultured alone (group A) andco-cultured with notochord cells (group B) (1 ∶ 1), and cell prol iferation and phenotype including proteoglycan and collagen II were evaluated. The cells in both groups were observed by the inverted microscope, and the survival rates of the primary and passage cells were detected by toluidine blue staining. The growth curves of the second passage cells in both groups were determined by MTT. Besides, the expressions of proteoglycan and collagen II of the primary and passage cells were examined by toluidine blue and immunocytochemistry staining. Results The notochord cells and chondroid cells were isolated and purified. With the diameter of 10-15 μm, the notochord cell had abundant intracytoplasmic vesicles, while the chondroid cell, with the diameter of 4-6 μm, had no intracytoplasmic vesicle. The cell survival rate was 89.0%-95.3% in group A and 91.3%-96.3% in group B. There was no significant difference between the same passages in both groups (P gt; 0.05). The co-cultured cells (group B) increased in cell prol iferation compared with the chondroid cells alone (group A) in repeated experiments. The cells in group A reached their logarithmic growth phase after 3-4 days of culture, while the cells in group B did after 2 days of culture. The cell prol iferation in group B was more than that in group A after 4-day culture (P lt; 0.05). The cocultured cells retained their phenotype for 5 passages, while parallel-cultured chondroid cells lost the expression of proteoglycan and collagen II after the third passage. Conclusion The notochord cells are conducive for the prol iferation and phenotypekeeping of the chondroid cells and may play a key role in preventing degeneration of the disc.