【Abstract】 Objective To develop a novel cartilage acellular matrix (CACM) scaffold and to investigate its performance for cartilage tissue engineering. Methods Human cartilage microfilaments about 100 nm-5 μm were prepared after pulverization and gradient centrifugation and made into 3% suspension after acellularization treatment. After placing the suspension into moulds, 3-D porous CACM scaffolds were fabricated using a simple freeze-drying method. The scaffolds were cross-l inked by exposure to ultraviolet radiation and immersion in a carbodiimide solution 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysucinimide. The scaffolds were investigated by histological staining, SEM observation and porosity measurement, water absorption rate analysis. MTT test was also done to assess cytotoxicity of the scaffolds. After induced by conditioned medium including TGF-β1, canine BMSCs were seeded into the scaffold. Cell prol iferation and differentiation were analyzed using inverted microscope and SEM. Results The histological staining showed that there are no chondrocytefragments in the scaffolds and that toluidine blue, safranin O and anti-collagen II immunohistochemistry staining werepositive. The novel 3-D porous CACM scaffold had good pore interconnectivity with pore diameter (155 ± 34) μm, 91.3% ± 2.0% porosity and 2 451% ± 155% water absorption rate. The intrinsic cytotoxicity assessment of novel scaffolds using MTT test showed that the scaffolds had no cytotoxic effect on BMSCs. Inverted microscope showed that most of the cells attached to the scaffold. SEM micrographs indicated that cells covered the scaffolds uniformly and majority of the cells showed the round or ell iptic morphology with much matrix secretion. Conclusion The 3-D porous CACM scaffold reserved most of extracellular matrix after thoroughly decellularization, has good pore diameter and porosity, non-toxicity and good biocompatibil ity, which make it a suitable candidate as an alternative cell-carrier for cartilage tissue engineering.
Objective To investigate the effect of collagen type I concentration on the physical and chemical properties of the collagen hydrogel, and to analyze the effect of different concentrations of collagen type I hydrogel on the phenotype and gene expression of the chondrocytes in vitro. Methods Three kinds of collagen hydrogels with concentrations of 12, 8, and 6 mg/ mL (C12, C8, and C6) were prepared, respectively. The micro-structure, compressive modulus, and swelling ratio of the hydrogels were measured and analyzed. The chondrocytes at 2nd passage were cocultured with three kinds of collagen hydrogels in vitro, respectively. After 1-day culture, the samples were stained with fluorescein diacetate (FDA) / propidium iodide (PI) and the cell activity was observed under confocal laser microscope. After 14-day culture, HE staining and toluidine blue staining were carried out to observe the histological morphology, and mRNA expressions of chondrocytes related genes (collagen type II, Aggrecan, collagen type I, collagen type X, Sox9) were determined by real-time fluorescent quantitative PCR. Results With the increase of collagen type I concentration from 6 to 12 mg/mL, the physical and chemical properties of the collagen hydrogels changed significantly: the fiber network became dense; the swelling ratios of C6, C8, and C12 were 0.260 ± 0.055, 0.358 ± 0.072, and 0.539 ± 0.033 at 192 hours, respectively, showing significant differences among 3 groups (P lt; 0.05); and the compression modulus were (4.86 ± 0.96), (7.09 ± 2.33), and (11.08 ± 3.18) kPa, respectively, showing significant differences among 3 groups (P lt; 0.05). After stained with FDA/PI, most cells were stained green, and few were stained red. The histological observation results showed that the chondrocytes in C12 hydrogels aggregated obviously with b heterochromia, chondrocytes in C8 hydrogels aggregated partly with obvious heterochromia, and chondrcytes in C6 hydrogels uniformly distributed with weak heterochromia. Real-time fluorescent quantitative PCR results showed that the mRNA expressions of collagen type II and Aggrecan were at the same level in C12, C8, and C6; the expressions of collagen type I, Sox9, and collagen type X were up-regulated with the increase of collagen type I hydrogels concentration, and the expressions were the highest at 12 mg/mL and were the lowest at 6 mg/mL, showing significant differences among 3 groups (P lt; 0.05). Conclusion Increasing the concentration of collagen hydrogels leads to better mechanical properties and higher shrink-resistance, but it may induce the up-regulation of cartilage fibrosis and hypertrophy related gene expression.
Objective Extracellular matrix is one of the focus researches of the adi pose tissue engineering. To investigate the appropriate method to prepare the porcine skeletal muscle acellular matrix and to evaluate the biocompatibility of the matrix. Methods The fresh skeletal muscle tissues were harvested from healthy adult porcine and were sl iced into2-3 mm thick sheets, which were treated by hypotonic-detergent method to remove the cells from the tissue. The matrix was then examined by histology, immunohistochemistry, and scanning electron microscopy. The toxic effects of the matrix were tested by MTT. Human adi pose-derived stem cells (hADSCs) were isolated from adi pose tissue donated by patients with breast cancer, and identified by morphology, flow cytometry, and differentiation abil ity. Then, hADSCs of passage 3 were seeded into the skeletal muscle acellular matrix, and cultured in the medium. The cellular behavior was assessed by calcein-AM (CA) and propidium iodide (PI) staining at 1st, 3rd, 5th, and 7th days after culturing. Results Histology, immunohistochemistry, and scanning electron microscopy showed that the muscle fibers were removed completely with the basement membrane structure; a large number of collagenous matrix presented as regular network, porous-like structure. The cytotoxicity score of the matrix was grade 1, which meant that the matrix had good cytocompatibil ity. The CA and PI staining showed the seeded hADSCs had the potential of spread and prol iferation on the matrix. Conclusion Porcine skeletal muscle acellular matrix has good biocompatibility and a potential to be used as an ideal biomaterial scaffold for adi pose tissue engineering.
Objective To review the research progress of articular cartilage scaffold materials and look into the future development prospects. Methods Recent literature about articular cartilage scaffold for tissue engineering was reviewed, and the results from experiments and clinical application about natural and synthetic scaffold materials were analyzed. Results The design of articular cartilage scaffold for tissue engineering is vital to articular cartilage defects repair. The ideal scaffold can promote the progress of the cartilage repair, but the scaffold materials still have their limitations. Conclusion It is necessary to pay more attention to the research of the articular cartilage scaffold, which is significant to the repair of cartilage defects in the future.
Objective To elucidate the latest research progress and application of tissue engineered meniscus. Methods The literature concerning the advance in tissue engineered meniscus was extensively reviewed, then closely-related issues including seed cells, scaffolds, and bioreactors were analyzed. Results With more and more attention being paid to meniscus tissue engineering, different approaches and strategies for seed cells, scaffolds, and bioreactors have contributed to the generation of meniscal constructs, which are capable of restoring meniscal lesions to some extent, but translating successes in basic science research to clinical application is still limited. Conclusion More research for the optimal combination of the appropriate cell source, the scaffold type, and the proper physical and chemical factors for the stimulation of cells differentiation into tissue with optimal phenotypes in tissue engineered meniscus is still in needed, but the overall future looks promising.
Objective To fabricate a nanohydroxyapatite-chitosan(nano-HA-CS) scaffold with high porosity by a simple and effective technique and to evaluate the physical and chemical properties and the cytocompatibility of the composite scaffold. Methods The threedimensional nano-HA-CS scaffolds with high porosity were prepared by the in situ hybridization-freeze-drying method. The microscopic morphology and components of the composite scaffolds were analyzed by the scanning electron microscopy (SEM), the transmission electron microscopy（TEM）, the X-ray diffraction（XRD）examination, and the Fourier transformed infrared spectroscopy（FTIR）. The calvarial osteoblasts were isolated from the neonatal Wistar rats. The serial subcultured cells (3rd passage) were respectively seeded onto the nanoHACS scaffold and the CS scaffold, and then were cocultured for 2, 4, 6 and 8 hours. At each time point,four specimens from each matrix were taken to determine the celladhesion rate. The cell morphology was observed by the histological staining and SEM. Results The macroporous nanoHACS scaffolds had a feature of high porosity with a pore diameter from 100 to 500 μm (mostly 400500 μm). The scaffolds had a high interval porosity; however, the interval porosity was obviously decreased and the scaffold density was increased with an increase in the contents of CS and HA. The SEM and TEM results showed that the nanosized HA was synthesized and was distributed on the pore walls homogeneously and continuously. The XRD and FTIR results showed that the HA crystals were carbonatesubstituded and not wellcrystallized. The cytocompatibility test showed that the seeded osteoblasts could adhere the scaffolds, proliferating and producing the extracellular matrix on the scaffolds. The adherence rate for the nanoHACS scaffolds was obviously higher than that for the pure CS scaffolds. Conclusion The nano-HA-CS scaffolds fabricated by the in situ hybridization-freeze-drying method have a good physical and chemical properties and a good cytocompatibility; therefore, this kind of scaffolds may be successfully used in the bone tissue engineering.
Objective To review the latest development in the research on the application of the electrostatic spinning technology in preparation of the nanometer high polymer scaffold. Methods The related articles published at home and abroad during the recent years were extensively reviewed and comprehensively analyzed. Results Micro/nano-structure and space topology on the surfaces of the scaffold materials, especially the weaving structure, were considered to have an important effect on the cell adhesion, proliferation, directional growth, and biological activation. The electrospun scaffold was reported to have a resemblance to the structure of the extracellular matrix and could be used as a promising scaffold for the tissue engineeringapplication. The electrospun scaffolds were applied to the cartilage, bone, blood vessel, heart, and nerve tissue engineering fields. Conclusion The nanostructured polymer scaffold can support the cell adhesion, proliferation, location, and differentiation，and this kind of scaffold has a considerable value in the tissue engineering field.
Objective To observe the adhesion and prol iferation of late endothel ial progenitor cells (EPCs) planted on nanoporous PLLA scaffold in vitro and to provide a new approach that optimizes tissue engineered material. Methods Male and female New Zealand rabbits (weight 2.5-3.0 kg) were used. Isolated late EPCs from rabbit peri pheral blood were cultured. Electrostatic spinning technique was adopted to prepare misal igned nanofibers, al igned nanofibers and super-al igned nanofibers, and low temperature plasma technique was appl ied to prepare misal igned membrane, al igned membrane and super-al igned membrane. After being divided into group A (cells only), B (misal igned membrane), C (normal membrane), D (al igned membrane) and E (super-al igned membrane), the primary late EPCs (1 × 105/mL) werecultured on scaffolds and MTT method was used to detect cell prol iferation abil ity at 3, 5, 7, 9, 11, 13, 15 and 17 days afterculture. After being divided into group A (misal igned membrane), B (normal membrane), C (al igned membrane) and D (superal igned membrane), precipitation method was appl ied to detect cell adhesion rate at 4, 12 and 24 hours after compound culture, and the morphologic changes of cells were observed at 4, 24 and 72 hours after compound culture. Results Fiber diameters in nanofibrous PLLA scaffolds were 300-400 nm, with a porosity rate of above 90%. At 3, 5, 7, 9, 11, 13, 15 and 17 days after culture, A value of each group was increased with time and the cells in each group grew well, showing there was no significant difference between group A and group B at each time point (P gt; 0.05 ); during the period of 7-15 days after culture, the difference between groups C, D and E and groups A and B was significant (P lt; 0.05). At 4 hours after compound culture, the adhesion rate of group A was superior to that of groups B, C and D (P lt; 0.05); at 12 and 24 hours after compound culture, the adhesion rate of groups B, C and D was remarkably higher than that of group A (P lt; 0.05); significant difference was noted in each group between the time point of 4 hours and the time point of 12 and 24 hours after compound culture (P lt; 0.05), but no significant difference between 12 hours and 24 hours was detected (P gt; 0.05). Morphology observation demonstrated that cells grew well on the scaffolds, the cells in groups A and B grew sporadically and disorderly, while the cells in groups C and D attached and al igned along fiber and prol iferated, with an excretion of ECM. Group D was better at maintaining cell morphology. Conclusion Al igned and superal igned nanofibers of PLLA scaffold can promote the adhesion and prol iferation of seed cells on the scaffold and maintain good cell morphology, which is an appropriate candidate scaffold material for blood vessel tissue engineering. Late EPCs is an ideal cell source for blood vessel tissue engineering.
ObjectiveTo investigate the influences of lactic acid (LA), the final degradation product of polylactic acid (PLA) on the prol iferation and osteoblastic phenotype of osteoblast-l ike cells so as to provide theoretical basis for bone tissue engineering. MethodsRos17/2.8 osteoblast-l ike cells were harvested and divided into 3 groups. In groups A and B, the cells were cultured with the medium containing 4, 8, 16, 22, and 27 mmol/L L-LA and D, L-LA, respectively. In group C, the cells were cultured with normal medium (pH7.4). The cell prol iferation was determined with MTT method after 1, 3, and 5 days. The relative growth ratio (RGR) was calculated, and the cytotoxicity was evaluated according to national standard of China. In addition, the alkal ine phosphatase (ALP) activity of cells cultured with medium containing 4 mmol/L L-LA (group A), 4 mmol/ L D, L-LA (group B), and normal medium (group C) after 1 and 5 days were detected with ALP kits, and the relative ALP ratio (RAR) was calculated; after 21 days, the calcium nodules were tested with von Kossa staining method, and were quantitatively analyzed. ResultsWhen LA concentration was 4 mmol/L, the mean RGR of both groups A and B were all above 80%, and the cytotoxic grades were grade 0 or 1, which meant non-cytotoxicity. When LA concentration was 8 mmol/L and 16 mmol/ L, groups A and B showed cytotoxicity after 5 days and 3 days, respectively. When LA concentration was above 22 mmol/L, cell prol iferations of groups A and B were inhibited evidently after 1-day culture. At each LA concentration, RGR of group A was significantly higher than that of group B at the same culture time (P<0.05) except those at 4 mmol/L after 1-day and 3-day culture. After 1 day, the RAR of group A was significantly higher than that of group B on 1 day (144.1%±3.2% vs. 115.2%±9.8%, P<0.05) and on 5 days (129.6%±9.8% vs. 78.2%±6.9%, P<0.05). The results of von Kossa staining showed that the black gobbets in group A were obviously more than those of groups B and C. The staining area of group A (91.2%±8.2%) was significantly higher than that of groups B (50.3%±7.9%) and C (54.2%±8.6%) (P<0.05). ConclusionThe concentration and composition of LA have significant effects on the cell proliferation and osteoblastic phenotype of osteoblast-l ike cells.
Objective To explore a novel nanometer biomaterial which could induce the regeneration of tooth tissues intell igently, and to evaluate the feasibil ity of using this kind of biomaterial as the scaffold for tooth tissue engineering by investigating the role it plays in tooth tissue engineering. Methods The scaffold for tooth tissue engineering containing recombinant human bone morphogenetic protein 2 (rhBMP-2) was prepared by mixing nanoscale β tricalcium phosphate (β-TCP)/collagen particles. Forty-six 8-10 weeks old specific pathogen free Sprague Dawley (SD)rats, including 34 females and 12 males, weighing 250-300 g, were involved in this study. Tooth germs were removed under a stereomicroscope from the mandible of newborn SD rat, then digested and suspended. Scanning electronic microscope (SEM), adhesion rate of cells, and MTT assay were used to evaluate the effects of the scaffold on the tooth germ cells cultured in vitro. The tissue engineered tooth germ which was constructed by tooth germ cells and scaffold was transplanted under SD rat’s kidney capsule as the experimental group (n=12); the tooth germ cells (cell-control group, n=12) or scaffold without cells (material-control group, n=4) were transplanted separately as control groups Specimens were harvested to perform general and histological observations at 4 and 8 weeks after transplantation. Results β-TCP/collagen showed a loose and porous appearance with soft texture and excellent hydrophil icity. Tooth germ cells grew well and could attach to the scaffold tightly 3 days after coculture. The adhesion rates of tooth germ cells were 27.20% ± 2.37%, 44.52% ± 1.87%, and 73.81% ± 4.15% when cocultured with scaffold for 4, 8, and 12 hours, respectively. MTT assay showed that the cell prol iferation status of experimental group was similar to that of the control group, showing no significant difference (P gt; 0.05). Some white calcified specimens could be harvested at 4-8 weeks after transplantation. At 4 weeks after transplantation some typical structures of dental cusp and enamel-dentin l ike tissues could be seen in the experimental group. Enamel-dentin l ike tissues also formed in some specimens of cell-control group, but they arranged irregularly. At 8 weeks after transplantation the enamel-dentin l ike tissue of experimental group exhibited a mature appearance and organized structure in comparison with that at 4 weeks. And mature enamel or dentin l ike tissue also could be seen in cell-control group. In contrast, there was no enamel or dentin l ike tissue in material-control group at 4 or 8 weeks after transplantation. Conclusion rhBMP-2 decorated β-TCP/collagen scaffold has good biocompatibil ity and can be used as a novel nanometer biomaterial, so it is a good choice in scaffolds for tooth tissue engineering.