This study investigated the early mechanical adaptability and osteogenic differentiation of mouse bone marrow mesenchymal stem cells (M-BMSCs) under micro-vibration stimulation (MVS). M-BMSCs were stimulated by MVS in vitro, cell proliferation, alkaline phosphatase (ALP) activity assay, and cytoskeleton were measured, and cell apoptosis was observed by flow cytometry. Early osteoblast-associated genes, runt-related transcription factor 2 (Runx2), Collagen Ⅰ (Col-Ⅰ) and ALP, were observed by RT-PCR and the activation of extracellular regulated protein kinases 1/2 (ERK1/2) was determined by Western blotting. The results showed that MVS had no significant effect on the proliferation of M-BMSCs. The early apoptosis was induced by mechanical stimulation (for one day), but the apoptosis was decreased after cyclic stimulation for 3 days. At the same time, MVS significantly accelerated the expression of F-actin protein in cytoskeleton, the synthesis of ALP and the ERK1/2 pathway, also up-regulated the expressions of Runx2, Col-Ⅰ and ALP genes. This study indicates that MVS could regulate cellular activity, alter early adaptive structure and finally promote the early osteogenic differentiation of M-BMSCs.
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.
【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 provide an ideal seed cell for tissue engineered urinary bladder and urethra by serially culturing canine smooth muscle cells from urinary bladder in vitro and compare biological characteristics of different passagesof cells. Methods Bladder smooth muscle cells of 12-month-old male dogs weighing 10-12 kg were isolated from adult dogs’ urinary bladders by collagenase and trypsin digestion and serially cultured in DMEM medium supplemented with 10% serum of newborn bovines. Morphology and prol iferation of the cells were observed and the serially-cultured cells were identified with the transmission electron microscope and immunohistochemistry. Results The cells appeared spindle in parallel rows when they grew to the degree of subconfluence, and showed the “peak-valley” structure under the inverted phase contrast microscope. The cells could be prol iferated serially to the 12th passage in vitro. The growth curve showed the cells before the 7th passage had the similar prol iferation characteristics and the growth cycle was about 40 hours. The TEM showed myofilament and the dense body in cytoplasm of smooth muscle cells. Smooth muscle actin was positive by immunohistochemical staining. After the 7th passage, the cells’ growth became slow, and myofilament and the dense body in cytoplasm vanished. Conclusion The canine smooth muscle cells from urinary bladder can be serially cultured in vitro and highly purified and largely prol iferated by the appropriate method. The cells before the 7th passage can be used as optimal seed cells for tissue engineered urinary bladder and urethra.