Abstract: Bone Marrow Stromal Cells (BMSCs) have chondrogenesis potential if chondrogenic environments or factors are provided. This study tests the hypothesis that chondrocytes can promote BMSC chondrogenesis at non-chondrogensis site. Porcine BMSCs and auricular chondrocytes were mixed at different ratios and 2.5×107 mixed cells were resuspended in 0.5 ml 30％ Pluronic, and then the mixture was injected into nude mice subcutaneously as experimental groups. Chondrocytes or BMSCs at the same cell number were mixed with 0.5 ml Pluronic and injected respectively as controls. 2.5×107 chondrocytes were mixed and injected as low concentration chondrocyte control. 8 weeks later, all specimens in experimental groups and chondrocyte group formed mature cartilage with abundant collagen II expression. Mature lacuna structures and metachromatic matrices were also observed in these specimens with the same level of GAG contents. Average wet weight of specimens in experimental groups was over 70% of that in chondrocyte group. In contrast, specimens in BMSC group showed mainly fibrous tissue. Only a small amount of cartilage was formed in specimens of low concentration chondrocyte group and the average wet weight was below 30% of that in chondrocyte group. These results demonstrate that chondrocytes can provide chondrogenic microenvironment and thus promote in vivo chondrogenesis of BMSCs at non-chondrogenesis sites. It also indicates that Pluronic is an ideal injectable biomaterial for cartilage tissue engineering.
Abstract: Objective: To compare biocompatibility, degradation, and mechanical properties of
polyglycolic acid (PGA) unwoven and woven fibers as scaffolding materials for tendon engineering in vitro. Methods: Three kinds of PGA fibers were included in this study. PGA raw material (Purac Co, Holland) was spun into single PGA filaments that were further twisted into woven fibers (PGA- 1). PGA filaments (Nantong Holycon, China) were twisted into woven fibers (PGA-2) as well.
PGA-1 and PGA-2 served as experimental groups 1 and 2, while unwoven PGA fibers (Albany Co, USA) served as control group. Three types of PGA fibers were made into cord-like scaffolds that mimic tendon shape and compared with each other for biocompatibility, degradation and biomechanical properties. Avian tenocytes were isolated from digital flexor tendon and expanded in vitro. Cells of the second passage were seeded onto the PGA scaffolds. In the first 2 weeks, the cell-
PGA constructs were in vitro cultured without tension and observed for cell adhesion and matrix production. The constructs were then cultured under dynamic loading in a bioreactor for another 2 weeks followed by gross and histological examinations. Results: PGA unwoven fibers have the median diameter of 10µm, while PGA-1 and PGA-2 fibers have the median diameters of 200µm and 60µm, respectively. Microscopy showed that tenocytes adhered well to all three types of PGA fibers in the first 10 days and produced abundant matrices. However, cells showed poor viability on PGA-2 fibers after 10 days, yet good viability on the other two PGA fibers over 2 weeks of observation period. H&E staining showed that there were viable cells and abundant matrices in the control and PGA-1 groups, but not in PGA-2 group after 4 weeks of in vitro culture. Additionally, PGA unwoven fibers degraded faster than woven fibers (PGA-1 and -2). Interestingly, the PGAtenocyte constructs formed tendon-like tissue after 4 weeks of in vitro culture grossly and histologically. Furthermore, mechanical test demonstrated that both PGA woven fibers had much higher tensile strength than unwoven fibers. Conclusion: Different PGA fibers have different biocompatibility with seeded tenocytes. PGA woven fibers could bear more intense mechanical loading and degrade slower than unwoven fibers, which is essential for in vitro generation of tendon tissue. Thus PGA woven fibers might serve as a proper form of scaffolding material for in vitro tendon engineering in a bioreactor.
Abstract: The purpose of this research is to find out the interaction between histological alterations and mechanical properties of engineered tendon implanted in situ. Defects of 0.5cm-1.0cm were made at deep flexor tendons by surgical procedure. Engineered tendons using degradable scaffolds polyglytic acid (PGA) mesh and tendon cells were implanted to repair the defects. Chickens were killed respectively at 2 weeks, 4 weeks, 6 weeks, and 8 weeks after surgery. The implants were
taken out for histological examination, biomechanical test, and collagen synthesis assay. The results showed that after surgery the PGA scaffolds degraded fast and took precedence of collagen synthesis. There were not enough amount and maturation of the collagen fibers of the new tendon at 2-8 weeks after surgery. The biomechanical properties of new tendons were less than those of the normal tendon. Therefore, it is necessary to construct engineered tendons with better degradation
rate of scaffolds and suitable biomechanical stimulation so that more collagen synthesis and better biomechanical properties of new tendons can be developed early after implantation.
Abstract: The objective of this study is to investigate the proliferation and differentiation of stromal cells derived from human fat tissues cultured on substrates with different surface properties. In addition, the similar investigation was performed for the cells proliferated in different concentrations of basic fibroblast growth factor (bFGF). The culture substrates include several polymer films with different water wettabilities, glass or a cell culture plate, and that coated with collagen type I or IV, gelatin, and bFGF. The proliferation profiles of cells were influenced by the type of culture substrates and the growth factor concentration. A larger number of cells proliferated was observed for substrates with the water wettability around 80o, while the cell number was significantly larger for every protein-coated substrate. The rate of cell proliferation became maximum in a bFGF concentration of 1,000 ng/mL. The bFGF concentration used for cell proliferation affected the differentiation profile of cells proliferated. The stromal cells proliferated in 1 ng/mL bFGF were osteogenically differentiated to the strongest and fastest extent among those in
other growth factor doses. The alkaline phosphatase (ALP) activity of cells increased with the increased cell number although the activity per cells was indentical, irrespective of the substrates type. The strongest adipogenic differentiation was observed for cells proliferated in 1,000 ng/mL bFGF and the differentiation induction was maintained for a long time period. No clear dependence
of the cell number on adipogenesis was observed. For chondrogenic differentiation, the bFGF concentration had no influence on the glycosaminoglycans (GAG) amount. These findings indicate that the proliferation and differentiation of human fat tissue-derived stromal cells are influenced by the culture substrate and the concentration of bFGF used for proliferation.
Abstract: A culture system that is capable of providing even and uniform distribution and
deposition of cells and extracellular matrix (ECM) is desired to enhance biological functions of the tissue-engineered artificial dermis (TEADs). For this purpose, we have developed a perfusion culture system that offers uniform exchange of nutrients and gases along the scaffold. Viability and effectiveness of the system were investigated by comparing biological and mechanical properties of TEADs. Results showed that the TEADs constructed by the perfusion culture system revealed significantly increased cell growth, ECM synthesis, and elastic modulus compared to those by the conventional static culture system. In addition, histological findings indicated that cells were more evenly distributed and ECM deposition increased in TEADs with the perfusion culture system. Therefore, it can be suggested that the perfusion culture system can constitute a more promising
approach for constructing the TEADs.
Abstract: A new method to fabricate porous chitosan nerve conduits with multi-channels was
described. A uniquely designed mold was composed of 7-50 stainless steel needles and a set of plastic pedestals. Porous or imperforate chitosan tubes with 2-5mm inner diameter and 0.2-1.0 mm wall thickness were made firstly. The chitosan tubes were injected with 3% chitosan gel. The stainless steel needles longitudinally perforated through the chitosan tubes filled with chitosan gel, and the plastic pedestals were used to fix the needles. Lyophilization was used to finish fabrication.
The diameter of channels was 0.2-0.4mm. Swelling property and biodegradability of
Multi-channeled chitosan conduits were investigated. Wright’s staining and scanning electron microscope (SEM) were used to observe spread and proliferation of Neuroblastoma cells (N2A, mouse) on the conduits. It is promising that the porous chitosan nerve conduits with multi-channels are used as nerve tissue engineering scaffolds in repair of peripheral nerve and spinal cord injuries.
Abstract: Recent study shows that endothelial progenitor cells (EPCs) and gene therapy technologies are effective strategies in the inhibition of stenosis and thrombus formation and improving the patency rate of the vascular graft in vivo. In this study, rat EPCs were cultured from bone marrow, and plated in fibronectin-coated plates with EBM-2 medium. Bone marrow mesenchymal stem cells (MSCs) were cultured with alpha minimum essential medium ( -MEM). After two weeks, EPCs
were immunohistochemically characterized using antibodies specific for endothelial cells. Retroviral vectors pMSCV-eNOS, pMSCV-tPA, pMSCV-LacZ and pMCSV-GFP were constructed. Retroviral particles were produced using packaging cell line 293T cells. Gene transfer was carried out by exposing cells to virus solution for 6 hours in the presence of 8µg/ml polybrene. For constructing vessels, MSCs and EPCs were seeded on fibronectin coated ePTFE graft in tissue culture condition for 2-4 weeks. The attachment and growth of cells were analyzed with scanning
electron microscopy (SEM). Our data showed that the EPCs expressed VEGF, Lectin BS-1, RECA-1, indicating they are endothelial lineage. The concentrated retroviral particles showed many folds higher transduction efficiency to NIH 3T3 cells than the commercial reagent Fugene. SEM data showed dense attachment of MSCs on the graft surface. MSCs/EPCs co-culture gave much better cell coverage on the graft than culture of EPCs alone.
Abstract: Fibrin is a natural polymer with excellent biocompatibility and widely used as a cell
delivery vehicle in tissue engineering. However, fibrin of low concentration is not able to promote cell growth and differentiation within a desired time because of contraction and biodegradation of cell-seeded matrices. In this study we investigated effects of combining fibrin with collagen on growth and osteogenic differentiation of bone marrow stromal cells (BMSCs). Rabbit BMSCs-populated fibrin hydrogels with or without collagen were fabricated and cultured by the free floating method for 4 weeks. The DNA content of fibrin/collagen matrix significantly increased the growth of BMSCs compared to the fibrin-only matrix at 2week. Alkaline phosphatase activity was significantly higher in the fibrin/collagen matrix (71.0 nmol of p-nitrophenol /min/disc) than the fibrin-only matrix (45.1 nmol of p-nitrophenol /min/disc). Deposition of calcium was not significantly different between two groups. Histological examination also revealed more matured organization and deposition of collagen fibers and more concentric calcium deposition in the
fibrin/collagen matrix compared to the fibrin-only matrix. These results indicated that
fibrin/collagen matrix could be more effective than fibrin alone in supporting growth and osteogenic differentiation of BMSCs.
Abstract: Bone marrow mesenchymal stem cells (BMSCs) possess a high replicative capacity and have the capacity to differentiate into various connective tissue cell types. With the advance in cell culture technique, the BMSCs have been induced to differentiate to osteoblastics linage. To improve the situation of non-union in posterior spinal fusion (PSF), tissue engineering approach to combine BMSCs supported by the calcium phosphate ceramics was applied in PSF and its effect
was investigated in the present study. Autologous BMSCs from 16-week-old rabbit tibiae were expanded and induced to differentiate into osteoblastic cells with defined medium and osteogenic supplement. Calcium phosphate ceramic (CPC) was used as the scaffold to deliver the cells to the standardized rabbit posterior spinal fusion model. The assessment of bone mineral and fusion mass volume was conducted at week 7 post-operation with quantitative computed tomography and
micro-computed tomography. When compared with control, the composite of BMSCs with CPC enhanced the fusion mass volume by 40% (p<0.05) and bone mineral content in the CPC was 7% (p=0.05) higher. Our study showed that additional BMSCs at the fusion site of PSF did provide extra resource for new bone formation and enhanced the fusion rate.