Objective To study the adhesion characteristic in vitrobetween porous biphasic calcium phosphate(BCP) nanocomposite and bone marrow mesenchymal stem cells (MSCs) that have been induced and proliferated. Methods MSCs obtained from SD ratbone marrow were in vitro induced and proliferated. After their osteoblastic phenotype were demonstrated, MSCs were seeded onto prepared porous BCP nanocomposite(experiment group)and common porous hydroxyapatite (control group). Their adhesion situation was analyzed by scanning electron microscope. The initial optimal cell seeding density was investigated between new pattern porous BCP nanocomposite and MSCs by MTT automated colormetric microassay method. Results The differentiation of MSCs to osteoblastic phenotype were demonstrated by the positive staining of mineralized node, alkaline phosphatase (ALP) and collagen typeⅠ, the most appropriate seeding density between them was 2×106／ml. The maximal number which MSCs could adhere to porous BCP nanocomposite was 1.28×107/cm3. Conclusion MSCs can differentiate to osteoblastic phenotype.The MSCs were well adhered to porous BCP nanocomposite.
Objective To comment on the recent advances of production and application of the bio-derived scaffold in the tissue engineered peripheral nerve. Methods The recent articles were systematically analyzed, and then the production methods of the bio-derived scaffold and its application to the tissue engineered peripheral nerve were evaluated and prospected. Results B iological tissues were processed by some methods to produce the bio-derived materials. These mat erials could maintain the structure and components of the tissues. Moreover, the immunogenicity of these materials was reduced. Conclusion Application of the bio-derived materials is a trend in the fabricating scaffold of the tissue en gineered peripheral nerve.
OBJECTIVE: To sum up the clinical results of bio-derived bone transplantation in orthopedics with tissue engineering technique. METHODS: From January 2000 to May 2002, 52 cases with various types of bone defect were treated with tissue engineered bone, which was constructed in vitro by allogeneous osteoblasts from periosteum (1 x 10(6)/ml) with bio-derived bone scaffold following 3 to 7 days co-culture. Among them, there were 7 cases of bone cyst, 22 cases of non-union or malunion of old fracture, 15 cases of fresh comminuted fracture of bone defect, 4 cases of spinal fracture and posterior route spinal fusion, 3 cases of bone implant of alveolar bone, 1 case of fusion of tarsotarsal joint. The total weight of tissue engineered bone was 349 g in all the cases, averaged 6.7 g in each case. RESULTS: All the cases were followed up after operation, averaged in 18.5 months. The wound in all the case healed by first intention, but 1 case with second intention. Bone union was completed within 3 to 4.5 months in 50 cases, but 2 cases of delayed union. Six cases were performed analysis of CD3, CD4, CD8, ICAM-1 and VCAM-1 before and after operation, and no obvious abnormities were observed. CONCLUSION: Bio-derived tissue engineered bone has good osteogenesis. No obvious rejection and other complications are observed in the clinical application.
Objective To locate sinoatrial node (SAN) in suckl ing pigs, to develop a rel iable method for isolation, purification and cultivation of SAN cells and to observe the compatibil ity of SAN cells and Col I fiber scaffold. Methods Five newborn purebred ChangBaiShan suckl ing pigs (male and female), aged less than 1-day-old and weighing 0.45-0.55 kg, wereused. Multi-channels electrophysiological recorder was appl ied to detect the original site of atrial waves. Primary SAN cells harvested from that area were cultured by the conventional culture method and the purification culture method including differential velocity adherent technique and 5-BrdU treatment, respectively. Atrial myocytes isolated from the left atrium underwent purified culture. Cell morphology, time of cell attachment, time of unicellular pulsation, and pulsation frequency were observed using inverted microscope. The purified cultured SAN cells (5 × 105 cells/mL) were co-cultured with prewetted Col I fiber scaffold for 5 days, and then the cells were observed by HE staining and scanning electron microscope (SEM). Results The atrial waves occurred firstly at the area of SAN. The purified cultured SAN cells were spindle, triangular, and irregular in morphology, and the spindle cells comprised the greatest proportion. Atrial myocytes were not spindle-shaped, but primarily triangular and irregular. The proportion of spindle cells in the conventional cultured SAN cells was decreased from 73.0% ± 2.9% in the purified cultured SAN cells, to 44.7% ± 2.3% (P lt; 0.01), and the proportion of irregular cells increased from 7.0% ± 1.7% in the purified cultrued SAN cells to 36.1% ± 2.6% (P lt; 0.01) . The proportion of the triangular cells in the purified and the conventional cultured SAN cells was 20.0% ± 2.1% and 19.2% ± 2.5%, respectively (P gt; 0.05). At 5 days after co-culture, HE staining displayed lots of SAN cells in Col I fiber scaffold, and SEM demonstrated conglobate adherence of the cells to the surface and lateral pore wall of scaffold, mutual connections of the cell processes, or attachment of cells to lateral pore wall of scaffold through pseudopodia. Conclusion With accurate SAN location, the purification culture method containing differential velocity adherent technique and 5-BrdU treatment can increase the proportion of spindle cells and is a rel iable method for the purification and cultivation of SAN cells. The SAN cells and Col I fiber scaffold have a good cellular compatibil ity.
Objective To compare the difference of preparing the acellular larynx scaffold between perfusion method and immersion method, and find better way to make acellular larynx scaffold for tissue engineering. Methods Twenty 6-month-old male New Zealand rabbits, weighing 2.0-2.5 kg, were divided into perfusion group (n=10) and immersion group (n=10) at random. All the larynxes were excised in a sterile fashion. The acellular larynx scaffold was obtained by perfusionmethod and immersion method respectively, and then comparative examinations were performed by the macroscopicview, histological view, scanning electron microscope (SEM), cartilage vital ity assay and toluidine blue staining. ResultsMacroscopic view showed that the larynxes perfused by sodium dodecyl sulphate (SDS) became transparent after 2 hoursof perfusion, but the larynxes immersed by SDS over 16 hours still appeared pink-white. Histology and SEM indicated thatcompared with immersion group, perfusion group showed better acellular effect, more ventages and collagen fibers wereretained, no intact cell or nuclei remained in acellular matrix and chondrocytes were still survival. The porosity was 85.39% ± 3.16% in perfusion group and 34.72% ± 4.51% in immersion group, showing significant difference (P lt; 0.01). The chondrocyte vital ity rate of perfusion group (86.93% ± 1.52%) was higher than that of immersion group (77.73% ± 1.66%), showing significant difference (P lt; 0.01). Toluidine blue staining showed that the chondrocyte heterochromaty was ber in perfusion group than that in immersion group. Conclusion Compared with immersion method, perfusion method is a better way to construct acellular larynx scaffold because it can achieve better acellular effect and retain chondrocyte vital ity at the greatest extent in the acellular larynx scaffold.
Objective To construct a new type of self-assembling peptide nanofiber scaffolds—RGDmx, and to study the cell compatibility of the new scaffolds and the proliferation and chondrogenic differentiation of precartilaginous stem cells(PSCs) in scaffolds. Methods PSCs were separated and purified from newborn Sprague Dawley rats by magnetic activated cell sorting and indentified by immunohistochemistry and immunofluorescent staining. The RGDmx were constructed by mixing KLD-12 and KLD-12-PRG at volume ratio of 1 ∶ 1. PSCs at passage 3 were seeded into the KLD-12 scaffold (control group) and RGDmx scaffold (experimental group). The proliferation of PSCs in 2 groups were observed with the method of cell counting kit (CCK) -8 after 1, 3, 7, and 14 days after culture. The RGDmx were constructed by mixing KLD-12-PRG and KLD-12 at different volume ratios of 0, 20%, 40%, 60%, 80%, and 100% and the prol iferation of PSCs was also observed. The complete chondrogenic medium (CCM) was used to induce chondrogenic differentiation of PSCs in different scaffolds. The differentiation of PSCs was observed by toluidine blue staining and RT-PCR assay. Results PSCs were separated and purified successfully, which were identified by immunohistochemistry and immunofluorescent staining methods. The results of CCK-8 showed that the absorbance (A) value in the experimental group increased gradually and reached the highest at 7 days; the A value in the experimental group was significantly higher than that in the control group at 7 days and 14 days (P lt; 0.05). Meanwhile, the A value in the RGDmx scaffold with a volume ratio of 40% was significantly higher than those in others (P lt; 0.05). After 14 days of induction culture with CCM, the toluidine blue staining results were positive in 2 groups; the results of RT-PCR showedthat the expression levels of collagen type II and the aggrecan in the experimental group were significantly higher than those in the control group (P lt; 0.05). Conclusion The self-assembling peptide nanofiber scaffold—RGDmx is an ideal scaffold for tissue engineer because it has good cell compatibility and more effective properties of promoting the differentiation of PSCs to chondrocytes.
Objective To establish a model for studying on mechanical responses of osteoblasts seeded in 3 dimensional(3D) scaffold. Methods Fifty pieces of bioderived cancellous bones, whose holes were 500 to 800 μm and density was 0.36 to 0.45g/cm3, were obtained as the scaffolds. They were cultured with the third passage suspension of Wistar rat. Twenty-four of the 50 scaffolds were constructed under apparent strain sine waveform with amplitude of 1 000 με, frequency of 3 Hz, and duration of 3 min/d, as experimental group. The other scaffolds were control group. After 3day coculture, osteoblasts were observed with scanning electron microscope. The proliferation of the osteoblasts was checked by MTT on scheduled date. Results Scanning electron microscopic observation showed that osteoblasts ttached and spread on the trabeculae, which presented the validity of the model under proper mechanical condition. Experiment showed that mechanical environment promoted theproliferation of osteoblasts. The observation of proliferation of osteoblasts showed that the quantity of osteoblasts in the experimental group was higher than that in the control group 1,4,8,12,16,20,24, and 28 days after culturing. Therewas significant difference between the two groups 12,16,20,24,and 28 days afterculturing(P＜0.05). Conclusion The establishment of the model can facilitate the study of mechanical responses of osteoblasts under different conditions.
OBJECTIVE: To prepare chitosan-gelatin/hydroxyapatite (CS-Gel/HA) composite scaffolds, and to investigate the influence of components and preparing conditions to their micromorphology. METHODS: The CS-Gel/HA composite scaffolds were prepared by phase-separation method. Micromorphology and porosity were detected by using scanning electron microscope and liquid displacement method respectively. RESULTS: Porous CS-Gel/HA composite scaffolds could be prepared by phase-separation method, and their density and porosity could be controlled by adjusting components and quenching temperature. CONCLUSION: The study suggests the feasibility of using CS-Gel/HA composite scaffolds for the transplantation of autogenous osteoblasts to regenerate bone tissue.
OBJECTIVE: To explore the possibility of detergent acellularized porcine heart valve serving as a scaffold for tissue engineering valve. METHODS: The porcine aortic valves were acellularized by use of trypsin-EDTA. Triton X-100, RNase and DNase treatment. Biomechanical characteristics of fresh valves and acellularized valve were tested; also fresh valves, acellularized valve and valves treated with method of bioprothetic treatment were implanted subcutaneously in rats; frequently seeded with bovine aortic endothelial cells(BAECs), and then cultured for 7 days. RESULTS: The acellularization procedure resulted in complete removal of the cellular components while the construction of matrix was maintained. The matrix could be successfully seeded with in vitro expanded BAECs, which formed a continuous monolayer on the surface. There is no significant difference of PGI2 secretion of BAECs between cells seeded onto the acellular leaflets and that onto the wells of 24-wells plate (P gt; 0.05). CONCLUSION: Acellularied porcine aortic valve can be applied as a scaffold to develop tissue engineering heart valve.
ObjectiveTo summarize the research progress of tissue engineered bile duct in recent years.MethodsThis paper summarized recently-published papers related to tissue-engineered bile duct on in vitro test platform, scaffold materials, acquisition methods of seed cells, and in vivo repair effectiveness after the fusion of seed cells and materials, in an attempt to review the basic and clinical application studies of tissue-engineered bile duct.ResultsTissue-engineered bile duct had been developing rapidly. At present, great progress had been made in the fields of in vitro test platform, scaffold materials, seed cells, and repair effectiveness in animal models. However, further study was still needed in terms of its clinical application. The external bile duct platform included 3D printing and biological simulation; in the aspect of scaffold material, apart from the progress of various artificial materials, acellular matrix was introduced; the selection of seed cells included the induction and differentiation of bile duct-derived stem cells, human bone marrow mesenchymal stem cells (hMSCs), hepatic oval cell (HOC), pluripotent stem cells (PSCs), and other stem cells; animal models of tissue-engineered bile ducts had also achieved good results in animals such as pigs and dogs.ConclusionThe development of tissue-engineered bile duct will promote the progress of fundamental in vitro studies on extrahepatic biliary tract diseases, thus introducing new options to the clinical treatment of extrahepatic biliary tract injuries.
