Shuo Yin

A comparative study of proliferation and osteogenic differentiation of adipose-derived stem cells on akermanite and β-TCP ceramics

This study investigated the in vitro effects of akermanite, a new kind of Ca-, Mg-, Si-containing bioceramic, on the attachment, proliferation and osteogenic differentiation of human adipose-derived stem cells (hASCs). Parallel comparison of the cellular behaviors of hASCs on the akermanite was made with those on beta-tricalcium phosphate (beta-TCP). Scanning electron microscope (SEM) observation and fluorescent DiO labeling were carried out to reveal the attachment and growth of hASCs on the two ceramic surfaces, while the quantitative assay of cell proliferation with time was detected by DNA assay. Osteogenic differentiation of hASCs cultured on the akermanite and beta-TCP was assayed by ALP expression and osteocalcin (OCN) deposition, which was further confirmed by Real-time PCR analysis for markers of osteogenic differentiation. It was shown that hASCs attached and spread well on the akermanite as those on beta-TCP, and similar proliferation behaviors of hASCs were observed on the two ceramics. Both of them exhibited good compatibility to hASCs with only minor cytotoxicity as compared with the tissue culture plates. Interestingly, the osteogenic differentiation of hASCs could be enhanced on the akermanite compared with that on the beta-TCP when the culture time was extended to approximately 10 days. Thus, it can be ascertained that akermanite ceramics may serve as a potential scaffold for bone tissue engineering.

A small diameter elastic blood vessel wall prepared under pulsatile conditions from polyglycolic acid mesh and smooth muscle cells differentiated from adipose-derived stem cells

Smooth muscle layer plays an important role in maintaining homeostasis of blood vessels, thus generating a functional smooth muscle layer is a prerequisite for successful construction of blood vessels via tissue-engineering approach. In this study, we investigated the feasibility of constructing an elastic vessel wall in small diameter (less than 6 mm) using smooth muscle cells (SMCs) differentiated from human adipose-derived stem cells (hASCs) under pulsatile stimulation in a bioreactor. With the induction of transforming growth factor-beta1 (TGF-beta1) and bone morphogenetic protein-4 (BMP4) in combination for 7 days, hASCs were found to acquire an SMC phenotype characterized by the expression of SMC-related markers including smooth muscle alpha actin (alpha-SMA), calponin, and smooth muscle myosin heavy chain (SM-MHC). The SMCs derived from hASCs were seeded in polyglycolic acid (PGA) unwoven mesh and the cell-scaffold complex were subjected to pulsatile stimulation in a bioreactor for 8 weeks. The vessel walls engineered under the dynamic stimulation for 8 weeks showed a dense and well-organized structure similar to that of native vessels. The differentiated hASCs with dynamic loading were found to maintain their SMC phenotype within 3-dimensional PGA scaffold with a high level of collagen deposition close to that of native ones. Vessels constructed in the static condition showed a loose histological structure with less expression of contractile proteins. More importantly, the engineered vessel under pulsatile stimulation exhibited significant improvement in biomechanical properties over that generated from static conditions. Our results demonstrated that hASCs can serve as a new cell source for SMCs in blood vessel engineering, and an elastic small-diameter vessel wall could be engineered by in vitro culture of SMC-differentiated hASCs on the PGA scaffold with matchable biomechanical strength to that of normal blood vessels under pulsatile stimulation.

The stimulation of osteogenic differentiation of human adipose-derived stem cells by ionic products from akermanite dissolution via activation of the ERK pathway.

Our previous study indicates that akermanite, a type of Ca-, Mg-, Si-containing bioceramic, can promote the osteogenic differentiation of hASCs. To elucidate the underlying mechanism, we investigated the effect of the extract from akermanite, on proliferation and osteogenic differentiation of hASCs. The original extract was obtained at 200 mg akermanite/ml LG-DMEM and further diluted with LG-DMEM. The final extracts were denoted as 1/2, 1/4, 1/8, 1/16, and 1/32 extracts based on the concentrations of the original extract. The LDH assay and live/dead stain were used to reveal the cytotoxicity of the different extracts on hASCs, while the DNA assay was carried out to quantitatively evaluate the proliferation of cells after being cultured with the extracts for 1, 3 and 7 days. Flow cytometry for cell cycle analysis was carried out on cells cultured in two media (GM and 1/2 extract) in order to further analyze the effect of the extract on cell proliferation behaviors. Osteogenic differentiation of hASCs cultured in the extracts was detected by ALP expression and calcium deposition, and further confirmed by real-time PCR analysis. It was shown that Ca, Mg and Si ions in the extract could suppress the LDH release and proliferation of hASCs, whereas promote their osteogenic differentiation. Such effects were concentration-dependent with the 1/4 extract (Ca 2.36 mM, Mg 1.11 mM, Si 1.03 mM) being the optimum in promoting the osteogenic differentiation of hASCs. An immediate increase in ERK was observed in cells cultured in the 1/4 extract and such osteogenic differentiation of hASCs promoted by released ions could be blocked by MEK1-specific inhibitor, PD98059. Briefly, Ca, Mg and Si ions extracted from akermanite in the concentrations of 2.36, 1.11, 1.03 mM, respectively, could facilitate the osteogenic differentiation of hASCs via an ERK pathway, and suppress the proliferation of hASCs without significant cytotoxicity.

Repair of articular cartilage defect in non-weight bearing areas using adipose derived stem cells loaded polyglycolic acid mesh

The current study was designed to observe chondrogenic differentiation of adipose derived stem cells (ASCs) on fibrous polyglycolic acid (PGA) scaffold stabilized with polylactic acid (PLA), and to further explore the feasibility of using the resulting cell/scaffold constructs to repair full thickness articular cartilage defects in non-weight bearing area in porcine model within a follow-up of 6 months. Autologous ASCs isolated from subcutaneous fat were expanded and seeded on the scaffold to fabricate ASCs/PGA constructs. Chondrogenic differentiation of ASCs in the constructs under chondrogenic induction was monitored with time by measuring the expression of collagen type II (COL II) and glycosaminoglycan (GAG). The constructs after being in vitro induced for 2 weeks were implanted to repair full thickness articular cartilage defects (8mm in diameter, deep to subchondral bone) in femur trochlea (the experimental group), while scaffold alone was implanted to serve as the control. Histologically, the generated neo-cartilage integrated well with its surrounding normal cartilage and subchondral bone in the defects of experimental group at 3 months post-implantation, whereas only fibrous tissue was filled in the defects of control group. Immunohistochemical and toluidine blue staining confirmed the similar distribution of COL II and GAG in the regenerated cartilage as the normal one. A vivid remolding process with post-operation time was also witnessed in the neo-cartilage as its compressive moduli increased significantly from 50.55% of the normal cartilage at 3 months to 88.05% at 6 months. The successful repair thus substantiates the potentiality of using chondrogenic induced ASCs and PGA/PLA scaffold for cartilage regeneration.

In vitro engineering of fibrocartilage using CDMP1 induced dermal fibroblasts and polyglycolide.

This study was designed to explore the feasibility of using cartilage-derived morphogenetic protein-1 (CDMP1) induced dermal fibroblasts (DFs) as seed cells and polyglycolide (PGA) as scaffold for fibrocartilage engineering. DFs isolated from canine were expanded and seeded on PGA scaffold to fabricate cell/scaffold constructs which were cultured with or without CDMP1. Proliferation and differentiation of DFs in different constructs were determined by DNA assay and glycosaminoglycan (GAG) production. Histological and immunohistochemical staining of the constructs after being in vitro cultured for 4 and 6 weeks were carried out to observe the fibrocartilage formation condition. The fibrocartilage-specific gene expression by cells in the constructs was analyzed by real-time PCR. It was shown that in the presence of CDMP1 the proliferation and GAG synthesis of DFs were significantly enhanced compared to those without CDMP1. Fibrocartilage-like tissue was formed in the CDMP1 induced construct after being cultured for 4 weeks, and it became more matured at 6 weeks as stronger staining for GAG and higher gene expression of collagen type II was observed. Since only weak staining for GAG and collagen type II was observed for the construct engineered without CDMP1, the induction effect on the fibrocartilage engineering can be ascertained when using DFs as seed cells. Furthermore, the potential of using DFs as seed cells to engineer fibrocartilage is substantiated and further study on using the engineered tissue to repair fibrocartilage defects is currently ongoing in our group.

Regeneration of dentin–pulp-like tissue using an injectable tissue engineering technique

An injectable tissue engineering method was developed for dentin–pulp complex regeneration using an injectable scaffold, crosslinked hyaluronic acid gel (HAG). A cell–scaffold composite composed of HAG, tooth bud-derived dental mesenchymal cells (DMCs), and transforming growth factor-β1 (TGF-β1) was prepared and injected subcutaneously into nude mice. Moreover, β-tricalcium phosphate (β-TCP) and polyglycolic acid (PGA) were chosen as control scaffolds for dentin–pulp regeneration. The suitability of injectable HAG for dentin–pulp complex engineering was further demonstrated in empty tooth slices and the pulp chambers of mini pigs. Histological and immunohistochemical staining was carried out to identify the distinctive tubular dentin and pulp structure, which was further confirmed by the detection of several dentinogenesis-related genes, DSPP, DMP-1, MEPE, and BSP. It was found that a recognizable dentin–pulp-like tissue with a typical well-organized dentinal tubular structure, columnar odontoblast-like cells, was successfully engineered using the injectable HAG scaffold within the subcutaneous area of nude mice, according to histological staining. High expression of the genes DSPP, DMP-1, MEPE and BSP in the above neo-tissue as well as positive immunohistochemical staining for dentin sialoprotein (DSP) confirmed the dentinal characteristics. No typical dentin- or pulp-like tissue was formed when PGA or β-TCP were used as scaffolds. The efficacy of this method was further demonstrated in empty tooth slices and pulp chambers of mini pigs that had been pretreated by the removal of total pulp and partial dentin. Through the successful delivery of DMCs and TGF-β1 by the injectable HAG scaffold, the destroyed dentin was vividly repaired along with the formation of pulp-like tissue. Hence the current strategy to engineer dentin–pulp complex can overcome the difficulty of specific anatomical arrangement of pulp and dentin with minimal invasion, finally leading to regained vitality, which is difficult to realize by the current clinical treatments.

Reconstructing spinal dura-like tissue using electrospun poly(lactide-co-glycolide) membranes and dermal fibroblasts to seamlessly repair spinal dural defects in goats

Many neuro- and spinal surgeries involving access to the underlying nervous tissue will cause defect of spinal dural mater, further resulting in cerebrospinal fluid leakage. The current work was thus aimed to develop a package which included two layers of novel electrospun membranes, dermal fibroblasts and mussel adhesive protein for repairing spinal dural defect. The inner layer is electrospun fibrous poly(lactide-co-glycolide) membrane with oriented microstructure (O-poly(lactide-co-glycolide)), which was used as a substrate to anchor dermal fibroblasts as seed cells to reconstitute dura-like tissue via tissue engineering technique. The outer layer is chitosan-coated electrospun nonwoven poly(lactide-co-glycolide) membrane (poly(lactide-co-glycolide)-chitosan). During surgery, the inner reconstituted tissue layer was first used to directly cover dura defects, while the outer layer was placed onwards with its marginal area tightly immobilized to the surrounding normal spinal dura aided by mussel adhesive protein. Efficacy of the current design was verified in goats with spinal dural defects (0.6 cm × 0.5 cm) in lumbar. It was shown that seamless and quick sealing of the defect area with the implants was realized by mussel adhesive protein. Guided tissue growth and regeneration in the defects of goats were observed when they were repaired by the current package. Effective cerebrospinal fluid containment and anti-adhesion of the regenerated tissue to the surrounding tissue could be achieved in the current animal model. Hence, it could be ascertained that the current package could be a favorite choice for surgeries involving spinal dural defects.