Fulin Chen

The performance of bone marrow mesenchymal stem cell--implant complexes prepared by cell sheet engineering techniques.

This study investigated the hypothesis that cell sheets composed of multilayered rabbit bone marrow derived mesenchymal stem cells (MSC) could be assembled with two kinds of implants (surface-modified titanium and zirconia) for the construction of a MSC-implant. The MSC sheets were harvested from culture flasks, wrapped around implants to construct the complexes, and then cultured in osteogenic medium. The layered cell sheets integrated well with implants and remained viable, with small mineralized nodules visible on the implant surfaces for up to four weeks after culture. Cells on the implants underwent classical in vitro osteogenic differentiation with an associated elevation of alkaline phosphatase activity and bone- and vascular-related protein expression. In vivo, two kinds of cell sheet-implant complexes were transplanted under the skin of SCID mice and cultured for eight weeks. For the MSC sheet titanium implant complex, histological examination revealed that new bone tissue that formed around implants followed a predominantly endochondral pathway, exhibiting histological markers of native bone; for the MSC sheet zirconia implant complex, however, intramembranous ossification appeared to occur on the surface of the zirconia implant, as observed with typical osteocytes embedded in dense matrix and accompanied by both microvessels and marrow cavities. These findings demonstrate that MSC-implants possessing osteogenic and vascularization abilities can be produced using cell sheet engineering techniques in conjunction with routine implant materials, which provide a novel technology to modify the implant surface.

A novel strategy to engineer small-diameter vascular grafts from marrow-derived mesenchymal stem cells.

Tissue-engineered blood vessels have mainly relied on endothelial cells (ECs), smooth muscle cells (SMCs), and biocompatible materials. However, long-term results have revealed several material-related failures, such as stenosis, thromboembolization, and the risk of infection. Furthermore, SMCs from elderly persons have reduced capacity in proliferation and collagen production. Mesenchymal stem cells (MSCs) have the ability to differentiate into multiple cell lineages, including osteoblasts, chondrocytes, ECs, and SMCs. In the current experiment, rabbit MSCs were cultured to form a cell sheet. A tissue-engineered vascular graft (TEVG) was fabricated by rolling the MSC sheet around a mandrel. The TEVG was implanted into a defect of the common carotid artery after it was examined macroscopically and microscopically. Hematoxylin and eosin staining showed that cell sheet was composed of five to seven layers of cells with the thickness of 40-50 µm. Results from the adhesion assay revealed that MSCs had similar antiplatelet adhesion property to ECs. Histological analysis of TEVGs showed that the layers of the cell sheet had fully fused in vitro. After implantation, TEVGs had excellent patency and integrated well with the native vessel. The structure of the TEVGs was similar to that of the native artery 4 weeks after implantation. Electron microscopy showed that the implanted TEVGs endothelialized. These results indicated that a completely biological TEVG could be assembled with autologous MSCs. These TEVGs are useful for revascularization in humans, which would reduce the occurrence of complications caused by foreign materials.

Novel strategy to engineer trachea cartilage graft with marrow mesenchymal stem cell macroaggregate and hydrolyzable scaffold.

Limited donor sites of cartilage and dedifferentiation of chondrocytes during expansion, low tissue reconstruction efficiency, and uncontrollable immune reactions to foreign materials are the main obstacles to overcome before cartilage tissue engineering can be widely used in the clinic. In the current study, we developed a novel strategy to fabricate tissue-engineered trachea cartilage grafts using marrow mesenchymal stem cell (MSC) macroaggregates and hydrolyzable scaffold of polylactic acid-polyglycolic acid copolymer (PLGA). Rabbit MSCs were continuously cultured to prepare macroaggregates in sheet form. The macroaggregates were studied for their potential for chondrogenesis. The macroaggregates were wrapped against the PLGA scaffold to make a tubular composite. The composites were incubated in spinner flasks for 4 weeks to fabricate trachea cartilage grafts. Histological observation and polymerase chain reaction array showed that MSC macroaggregates could obtain the optimal chondrogenic capacity under the induction of transforming growth factor-beta. Engineered trachea cartilage consisted of evenly spaced lacunae embedded in a matrix rich in proteoglycans. PLGA scaffold degraded totally during in vitro incubation and the engineered cartilage graft was composed of autologous tissue. Based on this novel, MSC macroaggregate and hydrolyzable scaffold composite strategy, ready-to-implant autologous trachea cartilage grafts could be successfully fabricated. The strategy also had the advantages of high efficiency in cell seeding and tissue regeneration, and could possibly be used in future in vivo experiments.

Engineering tubular bone using mesenchymal stem cell sheets and coral particles

The development of bone tissue engineering has provided new solutions for bone defects. However, the cell-scaffold-based approaches currently in use have several limitations, including low cell seeding rates and poor bone formation capacity. In the present study, we developed a novel strategy to engineer bone grafts using mesenchymal stem cell sheets and coral particles. Rabbit bone marrow mesenchymal stem cells were continuously cultured to form a cell sheet with osteogenic potential and coral particles were integrated into the sheet. The composite sheet was then wrapped around a cylindrical mandrel to fabricate a tubular construct. The resultant tubular construct was cultured in a spinner-flask bioreactor and subsequently implanted into a subcutaneous pocket in a nude mouse for assessment of its histological characteristics, radiological density and mechanical property. A similar construct assembled from a cell sheet alone acted as a control. In vitro observations demonstrated that the composite construct maintained its tubular shape, and exhibited higher radiological density, compressive strength and greater extracellular matrix deposition than did the control construct. In vivo experiments further revealed that new bone formed ectopically on the composite constructs, so that the 8-week explants of the composite sheets displayed radiological density similar to that of native bone. These results indicate that the strategy of using a combination of a cell sheet and coral particles has great potential for bone tissue engineering and repairing bone defects.

Immunization with FSHβ fusion protein antigen prevents bone loss in a rat ovariectomy-induced osteoporosis model.

Osteoporosis, a metabolic bone disease, threatens postmenopausal women globally. Hormone replacement therapy (HTR), especially estrogen replacement therapy (ERT), is used widely in the clinic because it has been generally accepted that postmenopausal osteoporosis is caused by estrogen deficiency. However, hypogonadal α and β estrogen receptor null mice were only mildly osteopenic, and mice with either receptor deleted had normal bone mass, indicating that estrogen may not be the only mediator that induces osteoporosis. Recently, follicle-stimulating hormone (FSH), the serum concentration of which increases from the very beginning of menopause, has been found to play a key role in postmenopausal osteoporosis by promoting osteoclastogenesis. In this article, we confirmed that exogenous FSH can enhance osteoclast differentiation in vitro and that this effect can be neutralized by either an anti-FSH monoclonal antibody or anti-FSH polyclonal sera raised by immunizing animals with a recombinant GST-FSHβ fusion protein antigen. Moreover, immunizing ovariectomized rats with the GST-FSHβ antigen does significantly prevent trabecular bone loss and thereby enhance the bone strength, indicating that a FSH-based vaccine may be a promising therapeutic strategy to slow down bone loss in postmenopausal women.

Chm-1 gene-modified bone marrow mesenchymal stem cells maintain the chondrogenic phenotype of tissue-engineered cartilage.

BackgroundMarrow mesenchymal stem cells (MSCs) can differentiate into specific phenotypes, including chondrocytes, and have been widely used for cartilage tissue engineering. However, cartilage grafts from MSCs exhibit phenotypic alternations after implantation, including matrix calcification and vascular ingrowth.MethodsWe compared chondromodulin-1 (Chm-1) expression between chondrocytes and MSCs. We found that chondrocytes expressed a high level of Chm-1. We then adenovirally transduced MSCs with Chm-1 and applied modified cells to engineer cartilage in vivo.ResultsA gross inspection and histological observation indicated that the chondrogenic phenotype of the tissue-engineered cartilage graft was well maintained, and the stable expression of Chm-1 was detected by immunohistological staining in the cartilage graft derived from the Chm-1 gene-modified MSCs.ConclusionsOur findings defined an essential role for Chm-1 in maintaining chondrogenic phenotype and demonstrated that Chm-1 gene-modified MSCs may be used in cartilage tissue engineering.

Directing the Differentiation of Parthenogenetic Stem Cells Into Tenocytes for Tissue-Engineered Tendon Regeneration

Uniparental parthenogenesis yields pluripotent stem cells without the political and ethical concerns surrounding the use of embryonic stem cells (ESCs) for biomedical applications. In the current study, we hypothesized that parthenogenetic stem cells (pSCs) could be directed to differentiate into tenocytes and applied for tissue‐engineered tendon. We showed that pSCs displayed fundamental properties similar to those of ESCs, including pluripotency, clonogenicity, and self‐renewal capacity. pSCs spontaneously differentiated into parthenogenetic mesenchymal stem cells (pMSCs), which were positive for mesenchymal stem cell surface markers and possessed osteogenic, chondrogenic, and adipogenic potential. Then, mechanical stretch was applied to improve the tenogenic differentiation of pMSCs, as indicated by the expression of tenogenic‐specific markers and an increasing COL1A1:3A1 ratio. The pSC‐derived tenocytes could proliferate and secrete extracellular matrix on the surface of poly(lactic‐co‐glycolic) acid scaffolds. Finally, engineered tendon‐like tissue was successfully generated after in vivo heterotopic implantation of a tenocyte‐scaffold composite. In conclusion, our experiment introduced an effective and practical strategy for applying pSCs for tendon regeneration. Stem Cells Translational Medicine 2017;6:196–208

A novel approach for the cryodesiccated preservation of tissue-engineered skin substitutes with trehalose.

Tissue-engineered skin (TES) holds great promise for wound healing in the clinic. However, optimized preservation methods remain an obstacle for its wide application. In this experimental work, we developed a novel approach to preserve TES in the desiccated state with trehalose. The uptake of trehalose by fibroblasts under various conditions, including the trehalose concentration, incubation temperature and time, was studied. The cell viability was investigated by the MTT assay and CFSE/PI staining after cryodesiccation and rehydration. TES was then prepared and incubated with trehalose, and the wound healing effect was investigated after desiccated preservation. The results showed that the optimized conditions for trehalose uptake by fibroblasts were incubation in 200 mM trehalose at 37 °C for 8 h. Cryodesiccated cells and TES maintained 37.55% and 28.31% viabilities of controls, respectively. Furthermore, cryodesiccated TES exhibited a similar wound healing effect to normal TES. This novel approach enabled the preservation and transportation of TES at ambient temperature with a prolonged shelf time, which provides great advantages for the application of TES.

Directing Parthenogenetic Stem Cells Differentiate into Adipocytes for Engineering Injectable Adipose Tissue

The selection of appropriate seed cells is crucial for adipose tissue engineering. Here, we reported the stepwise induction of parthenogenetic embryonic stem cells (pESCs) to differentiate into adipogenic cells and its application in engineering injectable adipose tissue with Pluronic F-127. pESCs had pluripotent differentiation capacity and could form teratomas that include the three primary germ layers. Cells that migrated from the embryoid bodies (EBs) were selectively separated and expanded to obtain embryonic mesenchymal stem cells (eMSCs). The eMSCs exhibited similar cell surface marker expression profiles with bone morrow mesenchymal stem cells (BMSCs) and had multipotent differentiation capacity. Under the induction of dexamethasone, indomethacin, and insulin, eMSCs could differentiate into adipogenic cells with increased expression of adipose-specific genes and oil droplet depositions within the cytoplasm. To evaluate their suitability as seed cells for adipose tissue engineering, the CM-Dil labelled adipogenic cells derived from eMSCs were seeded into Pluronic F-127 hydrogel and injected subcutaneously into nude mice. Four weeks after injection, glistering and semitransparent constructs formed in the subcutaneous site. Histological observations demonstrated that new adipose tissue was successfully fabricated in the specimen by the labelled cells. The results of the current study indicated that pESCs have great potential in the fabrication of injectable adipose tissue.

Development and evaluation of a sandwich ELISA method for the detection of human CD306.

CD306, also known as soluble leukocyte-associated immunoglobulin-like receptor-2 (LAIR-2), is a member of an immunoglobulin superfamily with the shared characteristic of an immunoglobulin-like C2-type domain. CD306 is speculated to be secretory and has 84% similarity with the extracellular domain of CD305, which binds to the same ligands as CD306. However, data on its distribution are absent due to the lack of an efficient method to detect it. In this study, we successfully cloned the cDNA of CD306 from the peripheral blood mononuclear cells (PBMCs) of patients with hemorrhagic fever with renal syndrome. The fusion proteins were expressed and purified, and three strains of monoclonal antibodies (mAbs) against CD306 were prepared and characterized. The sandwich ELISA for detecting CD306 was established and optimized with sensitivity up to 15 pg/ml, and the assay showed high specificity for the detection of CD306. With this method, a right skewed frequency distribution of CD306 in the sera of healthy subjects was determined. The concentrations of CD306 in sera and urine were detected in patients with different diseases. Aberrantly high levels of CD306 were found in the sera of pregnant women and patients with inflammation and rheumatic heart disease and in the urine of pregnant women. Meanwhile, there was a positive correlation between CD306 and soluble CD305 expression and secretion levels in sera and urine samples from patients, and both proteins inhibited CD305-mediated immunosuppressive functions. Our results demonstrate that CD306 represents a potentially useful predictor for disease diagnosis and that the method developed has potential for clinical application.