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.
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.
ObjectiveTo explore the morphological and functional features of tissue engineered composite constructed with bone mesenchymal stem cells (BMSCs) as seeding cells, thermosensitive collagen hydrogel (TCH) and poly-L-lactic acid (PLLA) as the extracellular matrix (ECM) scaffolds in the dynamic culture system.
MethodsBMSCs were separated from long bones of Fischer344 rat, and cultured; and BMSCs at the 3rd generation were seeded on the ECM scaffold constructed with braided PLLA fiber and TCH. The BMSCs-ECM scaffold composite was cultured in the dynamic culture system which was designed by using an oscillating device at a frequency of 0.5 Hz and at swing angle of 70° (experimental group), and in the static culture system (control group) for 7 days. The general observation and scanning electron microscopy (SEM) observation were performed; total DNA content was measured at 0, 1, 3, and 7 days.
ResultsPLLA was surrounded by collagen to form translucent gelatiniform in 2 groups; and compact membrane developed on the surface of PLLA. SEM observation showed that BMSCs had high viability and were fusiform in shape with microvilli on the surface of cells, and arranged in line; collagen and cells filled in the pores of PLLA fiber in the experimental group. The cells displayed a flat shape on the surface; there were less cells filling in the pores of PLLA fiber in the control group. At 1, 3, and 7 days, total DNA content in the experimental group was significantly higher than that in control group (P < 0.05). The total DNA content were increased gradually with time in 2 groups, showing significant difference between at 0 day and at 7 days (P < 0.05).
ConclusionThe ECM constructed with TCH and PLLA has good biocompatibility. The dynamic cultivation system can promote the cell proliferation, distribution, and alignment on the surface of the composite, so it can be used for tissue engineered composite in vitro.
Objective To study the mechanism of ectopic osteogenesis of nacre/Polylactic acid (N/P) artificial bone combined with allogenic osteoblasts, and to explore the possibility as a scaffold material of bone tissue engineering. Methods The allogenic- osteoblasts seeded onto N/P artificial bone were co-cultured in vivo 1 week.The N/P artificial bone with allogenic osteoblasts were implanted subcutaneously into the left back sites of the New Zealand white rabbits in the experimental group and the simple N/P artificial bone into the right ones in the control group. The complexes were harvested and examined by gross observation, histologic analysis and immunohistochemical investigation 2, 4 and 8 weeks after implantation respectively.Results In experimental group, the osteoid formed after 4 weeks, and the mature bone tissue withbone medullary cavities formed after 8 weeks; but in control group there was nonew bone formation instead of abundant fibrous tissue after 4 weeks, and more fibrous tissue after 8 weeks.Conclusion N/P artificial bone can be used as an optical scaffold material of bone tissue engineering.
ObjectiveTo summarize the research progress of several three-dimensional (3-D) printing scaffold materials in bone tissue engineering.
MethodThe recent domestic and international articles about 3-D printing scaffold materials were reviewed and summarized.
ResultsCompared with conventional manufacturing methods, 3-D printing has distinctive advantages, such as enhancing the controllability of the structure and increasing the productivity. In addition to the traditional metal and ceramic scaffolds, 3-D printing scaffolds carrying seeding cells and tissue factors as well as scaffolds filling particular drugs for special need have been paid more and more attention.
ConclusionsThe development of 3-D printing porous scaffolds have revealed new perspectives in bone repairing. But it is still at the initial stage, more basic and clinical researches are still needed.
Objective To review the appl ication of electrospinning in preparation of tendon tissue engineered scaffolds, to describe its appl ication effect and prospects. Methods Recent l iterature was extensively reviewed and summarized from various aspects, concerning the appl ication of electrospinning in preparing tendon tissue engineered scaffolds. Results Because of its huge surface and high porosity, the electrospun fibers prepared by electrospinning technology have been widely used in the manufacture of tendon tissue engineered scaffolds in recent years. A variety of materials, including polylactic acid, have been successfully electrospun into various types of tendon tissue engineered scaffolds, and goodresults in the repair of tendon defect were achieved. Conclusion The electrospinning technology has provide a new way for the preparation of the tendon tissue engineered scaffolds, with the perfection of the technology they will have broad application prospects in the field of tendon tissue engineering.
Objective Mechanical stimulation and inductive factors are both crucial aspects in tissue engineered cartilage. To evaluate the effects of mechanical stimulation combined with inductive factors on the differentiation of tissue engineered cartilage. Methods Bone marrow mesenchymal stem cells (BMSCs) were isolated from newborn porcine (aged7 days and weighing 3-6 kg) and expanded in vitro. The BMSCs at passage 2 were seeded onto a scaffold of poly (lactic-coglycol ic acid) (PLGA) in the concentration of 5 × 107/mL to prepare cell-scaffold composite. Cell-scaffold composites were cultivated in a medium with chondrocyte-inducted factors (group A), in a vessel with mechanic stimulating only (group B), or mechanic stimulating combined with chondrocyte-inducted factors (group C) (parameters of mechanics: 1 Hz, 0.5 MPa, and 4 hours/day). Cell-scaffold composite and auto-cartilage served as positive control (group D) and negative control (group E), respectively. After 4 weeks of cultivation, the thickness, elastic modulus, and glycosaminoglycan (GAG) content of composites were measured. Additionally, BMSCs chondrogenic differentiation was assessed via real-time fluorescent quantitative PCR, immunohistochemistry, and histological staining. Results The thickness, elastic modulus, and maximum load in group C were significantly higher than those in groups A and B (P lt; 0.05). In groups A, B, and C, cartilage lacuna formation, GAG expression, and positive results for collagen type II were obsersed through HE staining, Safranin-O staining, and immunohistochemistry staining. The dyeing depth was deeper in group A than in group B, and in group C than in groups A and B; group C was close to group E. The GAG content in group C was significantly higher than that in groups A and B (P lt; 0.05). Real-time fluorescent quantitative PCR revealed that mRNA expressions of collagen type I, collagen type II, and GAG in group C were significantly higher than those in groups A and B (P lt; 0.05), and in group A than in group B (P lt; 0.05). Conclusion Mechanical stimulation combined with chondrocyte inductive factors can enhance the mechanical properties of the composite and induce higher expression of collagen and GAG of BMSCs.
ObjectiveTo review the research progress of the tissue engineering technique in the esophageal defect repair and reconstruction.
MethodsThe recently published clinical and experimental literature at home and abroad on the scaffold materials and the seeding cells used in the tissue engineered esophageal reconstruction was consulted and summarized.
ResultsA large number of basic researches and clinical applications show that the effect of the tissue engineered esophagus is close to the autologous structure and function of the esophagus and it could be used for the repair of the esophageal defect. However, those techniques have a long distance from the clinical application and need an acknowledged rule of technology.
ConclusionTissue engineering technique could provide an innovative theory for the esophageal defect reconstruction, but its clinical application need further research.
Objective To introduce the materials, preparative technique and endothel ial ization modification of scaffold. Methods The recent original articles about vascular tissue engineering were extensively reviewed and analyzed. Results The materials including natural materials, biodegradable polymers and composite materials were studied in the field of scaffold. The ways of casting, cell self-assembly, gel spinning and electrospinning were appl ied to prepare the scaffold of vascular tissue engineering. The modification of scaffold was one of the most important elements for vascular tissue engineering. Conclusion The recent researchs about scaffold of vascular tissue engineering focus on composite material and electrospinning, the modification of scaffold can improve the abil ity of adhesion to endothel ial cells.
OBJECTIVE: To study the feasibility of calcium polyphosphate fiber (CPPF) as the scaffold material of tendon tissue engineering. METHODS: CPPF (15 microns in diameter) were woven to form pigtail of 3 mm x 2 mm transverse area; and the tensile strength, porous ratio and permeability ratio were evaluated in vitro. Tendon cells (5 x 10(4)/ml) derived from phalangeal flexor tendon of SD rats were co-culture with CPPF scaffold or CPPF scaffold resurfaced with collagen type-I within 1 week. The co-cultured specimens were examined under optical and electric scanning microscope. RESULTS: The tensile strength of CPPF scaffolds was (122.80 +/- 17.34) N; permeability ratio was 61.56% +/- 14.57%; and porous ratio was 50.29% +/- 8.16%. CPPF had no obvious adhesive interaction with tendon cells, while CPPF of surface modified with collagen type-I showed good adhesive interaction with tendon cells. CONCLUSION: The above results show that CPPF has some good physical characteristics as scaffold of tendon tissue engineering, but its surface should be modified with organic substance or even bioactive factors.
