Objective To prepare the silk fibroin microcarrier loaded with clematis total saponins (CTS) (CTS-silk fibroin microcarrier), and to investigate the effect of microcarrier combined with chondrocytes on promoting rabbit knee articular cartilage defects repair. Methods CTS-silk fibroin microcarrier was prepared by high voltage electrostatic combined with freeze drying method using the mixture of 5% silk fibroin solution, 10 mg/mL CTS solution, and glycerin. The samples were characterized by scanning electron microscope and the cumulative release amount of CTS was detected. Meanwhile, unloaded silk fibroin microcarrier was also prepared. Chondrocytes were isolated from knee cartilage of 4-week-old New Zealand rabbits and cultured. The 3rd generation of chondrocytes were co-cultured with the two microcarriers respectively for 7 days in microgravity environment. During this period, the adhesion of chondrocytes to microcarriers was observed by inverted phase contrast microscope and scanning electron microscope, and the proliferation activity of cells was detected by cell counting kit 8 (CCK-8), and compared with normal cells. Thirty 3-month-old New Zealand rabbits were selected to make bilateral knee cartilage defects models and randomly divided into 3 groups (n=20). Knee cartilage defects in group A were not treated, and in groups B and C were filled with the unloaded silk fibroin microcarrier-chondrocyte complexes and CTS-silk fibroin microcarrier-chondrocyte complexes, respectively. At 12 weeks after operation, the levels of matrix metalloproteinase 9 (MMP-9), MMP-13, and tissue inhibitor of MMP 1 (TIMP-1) in articular fluid were detected by ELISA. The cartilage defects were collected for gross observation and histological observation (HE staining and toluidine blue staining). Western blot was used to detect the expressions of collagen type Ⅱ and proteoglycan. The inflammatory of joint synovium was observed by histological staining and inducible nitric oxide synthase (iNOS) immunohistochemical staining. Results The CTS-silk fibroin microcarrier was spherical, with a diameter between 300 and 500 μm, a porous surface, and a porosity of 35.63%±3.51%. CTS could be released slowly in microcarrier for a long time. Under microgravity, the chondrocytes attached to the surface of the two microcarriers increased gradually with the extension of culture time, and the proliferation activity of chondrocytes at 24 hours after co-culture was significantly higher than that of normal chondrocytes (P<0.05). There was no significant difference in proliferation activity of chondrocytes between the two microcarriers (P>0.05). In vivo experiment in animals showed that the levels of MMP-9 and MMP-13 in group C were significantly lower than those in groups A and B (P<0.05), and the level of TIMP-1 in group C was significantly higher (P<0.05). Compared with group A, the cartilage defects in groups B and C were filled with repaired tissue, and the repaired surface of group C was more complete and better combined with the surrounding cartilage. Histological observation and Western blot analysis showed that the International Cartilage Repair Scoring (ICRS) and the relative expression levels of collagen type Ⅱ and proteoglycan in groups B and C were significantly better than those in group A, and group C was significantly better than group B (P<0.05). The histological observation showed that the infiltration of synovial inflammatory cells and hyperplasia of small vessels significantly reduced in group C compared with groups A and B. iNOS immunohistochemical staining showed that the expression of iNOS in group C was significantly lower than that in groups A and B (P<0.05).Conclusion CTS-silk fibroin microcarrier has good CTS sustained release effect and biocompatibility, and can promote the repair of rabbit cartilage defect by carrying chondrocyte proliferation in microgravity environment.
Objective To review the recent progress of the researches in the field of cartilage tissue engineering, and to discuss the challenges in construction of tissue engineered cartilage. Methods Literature related with cartilage tissue engineering was reviewed and analyzed. Results Some techniques have been appl ied in cl inical. As far as the seeding cells, induced pluripotent stem cells have attracted much more attention. Current strategies of scaffold designing are trying to imitate both component and structure of natural extracellular matrix. Cartilage regeneration through the autologous cell homing technique el iminate the transplantation of exotic cells and has become the hot topic. Conclusion Successful treatment of the damaged cartilage using tissue engineering method will depend on the advances of stem cell technology development, biomimetic scaffolds fabrication and proper appl ication of growth factors.
ObjectiveTo summarize the progress of the roles and mechanisms of various types of stem cell-based treatments and their combination therapies in both animal studies and clinical trials of lymphedema. MethodsThe literature on stem cell-based treatments for lymphedema in recent years at home and abroad was extensively reviewed, and the animal studies and clinical trials on different types of stem cells for lymphedema were summarized.ResultsVarious types of stem cells have shown certain effects in animal studies and clinical trials on the treatment of lymphedema, mainly through local differentiation into lymphoid endothelial cells and paracrine cytokines with different functions. Current research focuses on two cell types, adipose derived stem cells and bone marrow mesenchymal stem cells, both of which have their own advantages and disadvantages, mainly reflected in the therapeutic effect of stem cells, the difficulty of obtaining stem cells and the content in vivo. In addition, stem cells can also play a synergistic role in combination with other treatments, such as conservative treatment, surgical intervention, cytokines, biological scaffolds, and so on. However, it is still limited to the basic research stage, and only a small number of studies have completed clinical trials. ConclusionStem cells have great transformation potential in the treatment of lymphedema, but there is no unified standard in the selection of cell types, the amount of transplanted cells, and the timing of transplantation.
Objective
To review the recent advances in the application of graphene oxide (GO) for bone tissue engineering.
Methods
The latest literature at home and abroad on the GO used in the bone regeneration and repair was reviewed, including general properties of GO, degradation performance, biocompatibility, and application in bone tissue engineering.
Results
GO has an abundance of oxygen-containing functionalities, high surface area, and good biocompatibility. In addition, it can promote stem cell adhesion, proliferation, and differentiation. Moreover, GO has many advantages in the construction of new composite scaffolds and improvement of the performance of traditional scaffolds.
Conclusion
GO has been a hot topic in the field of bone tissue engineering due to its excellent physical and chemical properties. And many problems still need to be solved.
ObjectiveTo study the effect of the loaded concentration gradient of nerve growth factor (NGF) immobilized conduit on rat peripheral nerve defect repair.
MethodsThe peripheral nerve conduits made of poly (ε-caprolactone)-block-poly (L-lactide-co-ε-caprolactone) were prepared with uniform loads or concentration gradient loads by combining differential absorption of NGF/silk fibroin (SF) coating, and the gradient of NGF was immobilized in the nerve conduits. ELISA method was used to exam the NGF release for 12 weeks in vitro. Twenty-four male Sprague Dawley rats (weighing, 220-250 g) were selected to establish the right sciatic nerve defect model (14 mm in length) and randomly divided into 4 groups according to repair methods. The transected nerve was bridged by a blank conduit without NGF in group A, by a conduit containing uniform loads of NGF in group B, by a conduit concentration gradient loads of NGF in group C, and by the autogenous nerve segment in group D. The gross observation, electrophysiological examination, histological observation, and transmission electron microscope observation were carried out to assess the nerve regeneration at 12 weeks after surgery.
ResultsThe cumulative release amount of NGF was (14.2±1.4) ng/mg and (13.7±1.3) ng/mg in gradient of NGF loaded conduits and uniform NGF loaded conduits respectively at 12 weeks, showing no significant difference (t=0.564, P=0.570). All the animals survived to completion of the experiment; plantar ulcers occurred at 4 days, which healed at 12 weeks; groups C and D were better than groups A and B in ulcerative healing. At 12 weeks after surgery, the compound muscle action potential of group A was significantly lower than that of groups B, C, and D (P<0.05), and group B was significantly lower than groups C and D (P<0.05), but no significant difference was found between groups C and D (P>0.05). The axon density of group C was significantly higher that of groups A, B, and D (P<0.05); group D was significantly higher than groups A, B, and C, and group C was significantly higher than groups A and B in the axon number, axon diameter, and area of muscle fiber (P<0.05); the thickness of myelin sheath of groups C and D was significantly larger than that of groups A and B (P<0.05), but no significant difference was found between groups C and D (P>0.05).
ConclusionGradient of NGF loaded nerve condnits for rat sciatic nerve defect has similar results to autogenous nerve, with a good bridge, which can promote the sciatic nerve regeneration, improve the myelinization of the regenerating nerve, and accelerate the function reconstruction of the regenerating nerve.
Objective
To evaluate the influence of PKH26 labeling on the biological function of the goat nucleus pulposus cells and the biological function of seeded cells in nude mice by in vivo imaging techonology.
Methods
Primary nucleus pulposus cells were isolated by enzymatic digestion from the nucleus pulposus tissue of the 1-year-old goat disc. The nucleus pulposus cells at passage 1 were labeled with PKH26 and the fluorescent intensity was observed under the fluorescence microscopy. The labeled cells were stained with toluidine blue and collagen type II immunocytochemistry. The cells viability and proliferation characteristics were assessed by trypan blue staining and MTT assay, respectively. Real-time fluorescent quantitative PCR was used to detect the gene expressions of collagen types I and II, and aggrecan. The fluorescent intensity and scope of the nucleus pulposus cells-scaffold composite in vivo for 6 weeks after implanting into 5 6-week-old male nude mice were measured by in vivo imaging technology.
Results
Primary nucleus pulposus cells were ovoid in cell shape, showing cluster growth, and the cells at passage 1 showed chondrocyte-like morphology under the inverted phase contrast microscope. The results of toluidine blue and collagen type II immunocytochemistry staining for nucleus pulposus cells at passage 1 were positive. The fluorescent intensity was even after labeling, and the cell viability was more than 95% before and after PKH26 labeling. There was no significant difference in cell growth curve between before and after labeling (P gt; 0.05). The real-time fluorescent quantitative PCR showed that there was no significant difference in gene expressions of collagen types I and II, and aggrecan between before and after labeling (P gt; 0.05). Strong fluorescence in nucleus pulposus cells-scaffold composite was detected and by in vivo imaging technology.
Conclusion
The PKH26 labeling has no effect on the activity, proliferation, and cell phenotype gene expression of the nucleus pulposus cells. A combination of PKH26 labeling and in vivo imaging technology can track the biological behavior of the cells in vivo.
Objective
To study the transfection and expression of pleiotrophin (Ptn) gene in mice adipose-derived stem cells (ADSCs) so as to provide a new approach for the treatment of ischemic injury.
Methods
ADSCs from clean inbred C57BL/6W mice (weighing, 15-20 g) were isolated and cultured in vitro. The cell surface markers (CD29 and CD44) of ADSCs were identified by flow cytometry. The ADSCs were transfected with plasmid pIRES2-LEGFPN1 (containing Ptn gene coding sequence) as experimental group (group A) and with plasmid pLEGFP-N1 (containing GFP gene coding sequence) as control group (group B). After ADSCs were transfected by different plasmids respectively, the cells containing Ptn gene were selected by G418 (the best selected concentration was 200 μg/mL), and the immunophenotype of the cells was identified by flow cytometry after transfection. Meanwhile, real-time fluorescence quantitative PCR and Western blot were used to analyse the expression levels of Ptn mRNA and PTN protein in selected cells.
Results
The mice ADSCs were isolated and cultured successfully in vitro. The positive rates of the cell surface markers CD29 and CD44 of ADSCs were 99.5% and 95.8%, respectively; the double positive rate of CD44 and CD29 was 93.6%. The positive rates of the cell surface markers CD29 and CD44 of ADSCs were 99.1% and 95.6%, respectively after transfection of Ptn gene; the double positive rate of CD44 and CD29 was 93.4%. The expression levels of Ptn gene and PTN protein in group A were significantly higher than those in group B (P lt; 0.05).
Conclusion
The ADSCs can be stablely transfected by Ptn gene, the transfected ADSCs can express PTN protein highly, which is a new idea for tissue engineering of vascular reconstruction.
Objective To prepare collagen-chitosan /nano-hydroxyapatite-collagen-polylactic acid (Col-CS/ nHAC-PLA) biomimetic scaffold and to examine its biocompatibility so as to lay the foundation for its application on the treatment of osteochondral defect. Methods PLA was dissolved in dioxane for getting final concentration of 8%, and the nHAC power was added at a weight ratio of nHAC to PLA, 1 ∶ 1. The solution was poured into a mold and frozen. CS and Col were dissolved in 2% acetum for getting the final concentrations of 2% and 1% respectively, then compounded at a weight ratio of CS to Col, 20 ∶ 1. The solution was poured into the frozen mold containing nHAC-PLA, and then biomimetic osteochondral scaffold of Col-CS/nHAC-PLA was prepared by freeze-drying. Acute systemic toxicity test, intracutaneous stimulation test, pyrogen test, hemolysis test, cytotoxicity test, and bone implant test were performed to evaluate its biocompatibility. Results Col-CS/nHAC-PLA had no acute systemic toxicity. Primary irritation index was 0, indicating that Col-CS/nHAC-PLA had very slight skin irritation. In pyrogen test, the increasing temperature of each rabbit was less than 0.6℃, and the increasing temperature sum of 3 rabbits was less than 1.3℃, which was consistent with the evaluation criteria. Hemolytic rate of Col-CS/nHAC-PLA was 1.38% (far less than 5%). The toxicity grade of Col-CS/nHAC-PLA was classified as grade I. Bone implant test showed that Col-CS/nHAC-PLA had good biocompatibility with the surrounding tissue. Conclusion Col-CS/ nHAC-PLA scaffold has good biocompatibility, which can be used as an alternative osteochondral scaffold.
ObjectiveTo observe transforming growth factor β3 (TGF-β3) gene expression and the chondrogenesis of bone marrow mesenchymal stem cells (BMSCs) after TGF-β3 gene is transfected into BMSCs of Diannan small-ear pig.
MethodsRecombinant adenovirus 5 (rAd5) was extracted as gene vector and packed into recombinant adenovirus rAd5-TGF-β3, double enzyme digestion and PCR identification were performed. BMSCs were isolated and cultured from bone marrow of 2-month-old Diannan small-ear pigs (weighing, 12-15 kg), and the 2nd generation of BMSCs were harvested for experiments. The experiments were divided into 3 groups. BMSCs were transfected with rAd5-TGF-β3 as experimental group and with empty vector as control group, and non-transfected BMSCs were used as blank control group. The transfection efficiency of exogenous gene was identified by flow cytometry, TGF-β3 protein expression by immunofluorescence and Western blot. The cell morphology of experimental group was observed by inverted phase contrast microscope, and the expression of collagen type II in each group was detected by Western blot.
ResultsThe rAd5-TGF-β3 recombinant adenovirus was successfully constructed and transfected into BMSCs. Green fluorescence was observed by immunofluorescence microscope. Flow cytometry test showed the best transfection at 72 hours (transfection efficiency of 84.86%). Immunofluorescence staining showed that the expression of TGF-β3 protein was obvious at 72 hours; Western blot showed that there was a TGF-β3 positive band with a relative molecular mass of 30×103, while the control group and blank control group had no positive band. Obvious chondrogenic differentiation was observed in the experimental group after transfection in vitro, while the control group and blank control group had no obvious chondrogenic differentiation. Western blot showed that there was collagen type II positive band with a relative molecular mass of 130×103 at 21 days after culture, while the control group and blank control group had no positive band.
ConclusionrAd5-TGF-β3 gene can be successfully transfected into BMSCs via adenovirus vectors, and stable expression of TGF-β3 protein can be observed, enhancing BMSCs differentiation into chondrocytes, which may provide an experimental basis for gene therapy of joint cartilage defects.
Small intestinal submucosa (SIS) is a natural decellularized extracellular matrix material. Due to its excellent biocompatibility, unique biomechanical properties and biological activity, it has been widely used as a scaffold in regenerative medicine. This article reviews the recent progress in the characterization and medical application of SIS respectively. The specific biological properties of the SIS, as well as its interaction with cells, are highlighted. Some of the SIS products and clinical cases are also reviewed and discussed.
