Objective To investigate the feasibility of a new kind of porous β tricalcium phosphate (β-TCP) as a scaffold for the bone tissue engineering Methods The inverted phase contrast microscope was used to observe the growth of the marrow mesenchymal stem cells (MSCs) in the experimentalgroup and the control group at 10 days.In the experimental group, the MSCs were cultured with β-TCP(3 mm×3 mm×3 mm) in the 24-hole cultivation board, and in the control to control group, only MSCs were cultivated. The scanning electron microscope was used to observe growth of MSCs at 6 days. Cultivated with β-TCP at 3, 6, 9, 12 days, the MTT assay was used to judge the biocompatibility. The cytotoxicity was analyzed with the method that used the different density(100%， 50%， 10%， 1%，0%) leaching liquor gained from β-TCP to raise MSCs. MSCs were induced into the osteoblasts and were mixed with β-TCP, and the composite was used to repair a large radius bone defect in the rabbit. The specimens were made at 2,6,12 weeks. The histology imageology, and the radionuclide bone scan were used to analyze the bone formation. Results Some MSCs had a good adherence 4 hours after MSCs were inoculated and had a complete adherence at 12 hours. The cells were shaped like polyangle, spindle or converge monolayer after 8-10 days. The cells in the two groups had no difference. The cell adhesion was good, when observed by the inverted phase contrast microscope and the scanning electron microscope at 6 days. MTT showed that the absorbance (A)was not statistically different between the experimental group and the control group (P＞0.05); the different density leaching liquor had no cytotoxicity at the different time points. Histology, X-ray, and CT tomograph showed that itcould repair the large radius bone defect in the rabbit and its in vivo degradationrate was the same as the bone formation rate. Conclusion The new porous β-TCP has a unique three dimensional (3D) stereochemical structure and superordinary physicochemical property, and so it is a good scaffold for the bone tissue engineering.
To review the structure and function of the calcified cartilage zone and its role in the pathogenesis of osteoarthritis (OA). Methods Recent l iterature about calcified zone was reviewed and analyzed in terms of architecture, composition, biomechanics, and biological function. Results Calcified zone has particular structure and material properties, and functions as a semi permeable membrane; chondrocytes in the calcified zone retain some characteristics of growth plate cells, which play a crucial role in cartilage function maintenance and pathogenesis of OA. Therefore, reconstructionof the calcified zone at osteochondral conjunction has become one of the hot research in the fields of interface tissue engineering. Conclusion It is necessary to pay more attention to calcified cartilage zone, which is important for both the treatment of OA and the preparation of tissue engineered osteochondral composite.
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 determine the short-term effectiveness of matrix-induced autologous chondrocyte implantation (MACI) for femoral trochlea cartilage injury. Methods A retrospective analysis was performed on the clinical data of 10 patients with femoral trochlea cartilage injury treated with MACI between June 2012 and October 2014. There were 6 males and 4 females, aged from 15 to 48 years (mean, 33 years). The left knee was involved in 3 cases and the right knee in 7 cases. Nine patients had a history of trauma, and 1 case suffered from osteochondritis dissecans. Combined injuries included meniscus injury in 1 case, anterior cruciate ligament injury in 3 cases, and lateral collateral ligament tear in 2 cases. The mean lesion depth was 2.80 mm (range, 2-7 mm), with the mean defect size of 84.85 mm2 (range, 28.26-153.86 mm2). The mean duration of definite diagnosis was 14 days (range, 5 days to 3 months). By using arthroscopic biopsy, 200-300 mg healthy articular cartilage at non weight-bearing area of the knee femoral trochlea was collected as a source of seed cells, which were isolated and cultured to prepare MACI membrane. The adhesion activity, growth rate, and mechanical properties of the chondrocytes on the Bio-gide collagen scaffold were evaluated. In addition, the stretch rate, tensile strength, and suture strength of scaffold were tested. MACI membrane was implanted after 2 weeks to 6 months. The visual analogou scale (VAS), Lysholm score, and Tegner movement level score at preoperation and last follow-up were used to assess the function. Results The MACI membrane was successfully prepared, and the human chondrocytes adhered and grew well on the Bio-gide collagen scaffold. Mechanical test showed that MACI membrane had the stretch rate of 65.27%, the tensile strength of 26.81 MPa, and the suture strength of 6.49 N, indicating good mechanical properties. MACI membrane was successfully implanted. The mean operation time was 58.5 minutes (range, 43-99 minutes), and the mean hospitalization time was 7 days (range, 6-15 days). All incisions healed well. Ten cases were followed up 9 to 16 months (mean, 12 months). Four cases underwent iliac bone graft surgery. The mean healing time was 14 weeks (range, 12-16 weeks). No complications of osteochondrolysis, knee pain, nerve and vascular injury, deep vein thrombosis, and knee adhesion occurred during follow-up. The VAS score, Lysholm score, and Tegner score at last follow-up were significantly improved when compared with preoperative scores (t=12.060,P=0.000;t=–9.200,P=0.000;t=–14.000,P=0.000). Conclusion MACI for femoral trochlea cartilage injury has good short-term effectiveness, with less injury and fast function recovery.
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 fundamental research and the experimental study in the nerve tissue engineering of self-assembl ing peptide nanofiber scaffold (SAPNS). Methods The l iterature concerning basic and experimental studies on SAPNS in the nerve tissue engineering was extensively reviewed. Results SAPNS can promote the neural stem cell adhesion,prol iferation, differentiation and neuron axon outward growth and extension, promote extracellular matrix synthesis and inhibit gl ial cell adhesion and differentiation, and simulate the environment of a cell in the body. Conclusion SAPNS is an ideal matrix material and provides a new way for the repair of nerve tissue injury.
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.