Haiyun Ma

Myogenic differentiation of human bone marrow mesenchymal stem cells on a 3D nano fibrous scaffold for bladder tissue engineering.

Current strategies for engineering bladder tissues include a bladder biopsy for in vitro cell expansion for use in reconstructive procedures. However, this approach cannot be used in patients with bladder cancer who need a complete bladder replacement. Bone marrow mesenchymal stem cells (BMSC) might be an alternative cell source to better meet this need. We investigated the effects of soluble growth factors, bladder extracellular matrix (ECM), and 3D dynamic culture on cell proliferation and differentiation of human BMSC into smooth muscle cells (SMC). Myogenic growth factors (PDGF-BB and TGF-beta1) alone, or combined either with bladder ECM or dynamic cultures, induced BMSC to express smooth muscle-specific genes and proteins. Either ECM or the dynamic culture alone promoted cell proliferation but did not induce myogenic differentiation of BMSC. A highly porous poly-l-lactic acid (PLLA) scaffold provided a 3D structure for maximizing the cell-matrix penetration, maintained myogenic differentiation of the induced BMSC, and promoted tissue remolding with rich capillary formation in vivo. Our results demonstrate that myogenic-differentiated BMSC seeded on a nano fibrous PLLA scaffold can be potentially used for cell-based tissue engineering for bladder cancer patients requiring cystoplasty.

Porous nanofibrous PLLA scaffolds for vascular tissue engineering

Tissue-engineered small-diameter vascular grafts are needed for patients requiring replacement of their injured coronary and below-the-knee vessels. Understanding the interactions between the scaffolds and implanted cells and therefore the phenotype control of smooth muscle cells (SMCs) is critical for constructing functional vascular grafts. In this study, the effect of nanofibrous (NF) poly-L-lactide (PLLA) scaffolds on phenotype control of human aortic smooth muscle cells (HASMCs) was investigated. A tubular NF PLLA scaffold for blood vessel regeneration was fabricated and cell seeding studies showed cell distribution throughout the scaffold. It was found that NF PLLA scaffolds preferentially supported contractile phenotype of HASMCs under the in vitro culture conditions, as evidenced by elevated gene expression level of SMCs contractile markers including smooth muscle myosin heavy chain, smoothelin and myocardin. In vivo subcutaneous implantation studies confirmed HASMCs differentiation in the implants. Taken together, the results showed promising application of the porous NF PLLA scaffolds for reconstruction of tissue-engineered vascular grafts.

Three-dimensional growth of iPS cell-derived smooth muscle cells on nanofibrous scaffolds

Induced pluripotent stem cells (iPSCs) have been considered as the major component for personalized regenerative medicine. However, the potential of iPSCs in constructing tissue-engineered (TE) blood vessels has not been exploited. In the present study, we generated mouse iPSCs with the combination of over-expression of 4 iPS factors and knock-down of p53 gene. The established iPSCs were then directed to differentiate into smooth muscle cells (SMCs) with the treatment of 10(-5) (M) all-trans retinoid acid (RA). The vehicle dimethyl sulfoxide (DMSO) treatment served as a spontaneous differentiation control. The differentiated cells were then cultured on three-dimensional (3D) macro-porous nanofibrous (NF) poly(L-lactide) (PLLA) scaffolds in vitro. Our data showed that the expression of SMC specific marker genes, including myocardin, smoothelin, SM22α and SMMHC, were higher for the group induced by RA than for the group treated by DMSO, while pluripotent marker gene expression was repressed by the RA-treatment. Upon subcutaneous implantation, the implanted cells maintained the SMC phenotype. In conclusion, the data suggest that iPSCs-derived SMCs can be an important cell source for personalized vascular tissue engineering applications.

Construction of Vascular Tissues with Macro-Porous Nano-Fibrous Scaffolds and Smooth Muscle Cells Enriched from Differentiated Embryonic Stem Cells

Vascular smooth muscle cells (SMCs) have been broadly used for constructing tissue-engineered blood vessels. However, the availability of mature SMCs from donors or patients is very limited. Derivation of SMCs by differentiating embryonic stem cells (ESCs) has been reported, but not widely utilized in vascular tissue engineering due to low induction efficiency and, hence, low SMC purity. To address these problems, SMCs were enriched from retinoic acid induced mouse ESCs with LacZ genetic labeling under the control of SM22α promoter as the positive sorting marker in the present study. The sorted SMCs were characterized and then cultured on three-dimensional macro-porous nano-fibrous scaffolds in vitro or implanted subcutaneously into nude mice after being seeded on the scaffolds. Our data showed that the LacZ staining, which reflected the corresponding SMC marker SM22α expression level, was efficient as a positive selection marker to dramatically enrich SMCs and eliminate other cell types. After the sorted cells were seeded into the three-dimensional nano-fibrous scaffolds, continuous retinoic acid treatment further enhanced the SMC marker gene expression level while inhibited pluripotent maker gene expression level during the in vitro culture. Meanwhile, after being implanted subcutaneously into nude mice, the implanted cells maintained the positive LacZ staining within the constructs and no teratoma formation was observed. In conclusion, our results demonstrated the potential of SMCs derived from ESCs as a promising cell source for therapeutic vascular tissue engineering and disease model applications.

Pore size directs bone marrow stromal cell fate and tissue regeneration in nanofibrous macroporous scaffolds by mediating vascularization

In the U.S., 30% of adults suffer joint pain, most commonly in the knee, which severely limits mobility and is often attributed to injury of cartilage and underlying bone in the joint. Current treatment methods such as microfracture result in less resilient fibrocartilage with eventual failure; autografting can cause donor site morbidity and poor integration. To overcome drawbacks in treatment, tissue engineers can design cell-instructive biomimetic scaffolds using biocompatible materials as alternate therapies for osteochondral defects. Nanofibrous poly (l-lactic acid) (PLLA) scaffolds of uniform, spherical, interconnected and well-defined pore sizes that are fabricated using a thermally-induced phase separation and sugar porogen template method create an extracellular matrix-like environment which facilitates cell adhesion and proliferation. Herein we report that chondrogenesis and endochondral ossification of rabbit and human bone marrow stromal cells (BMSCs) can be controlled by scaffold pore architecture, particularly pore size. Small-pore scaffolds support enhanced chondrogenic differentiation in vitro and cartilage formation in vivo compared to large-pore scaffolds. Endochondral ossification is prevented in scaffolds with very small pore sizes; pore interconnectivity is critical to promote capillary ingrowth for mature bone formation. These results provide a novel strategy to control tissue regenerative processes by tunable architecture of macroporous nanofibrous scaffolds. STATEMENT OF SIGNIFICANCE: Progress in understanding the relationship between cell fate and architectural features of tissue engineering scaffolds is critical for engineering physiologically functional tissues. Sugar porogen template scaffolds have uniform, spherical, highly interconnected macropores. Tunable pore-size guides the fate of bone marrow stromal cells (BMSCs) towards chondrogenesis and endochondral ossification, and is a critical design parameter to mediate neotissue vascularization. Preventing vascularization favors a chondrogenic cell fate while allowing vascularization results in endochondral ossification and mineralized bone formation. These results provide a novel strategy to control tissue regenerative processes by tunable architecture of macroporous nanofibrous scaffolds.