Kuei-Chang Li

Recent progresses in gene delivery-based bone tissue engineering

Gene therapy has converged with bone engineering over the past decade, by which a variety of therapeutic genes have been delivered to stimulate bone repair. These genes can be administered via in vivo or ex vivo approach using either viral or nonviral vectors. This article reviews the fundamental aspects and recent progresses in the gene therapy-based bone engineering, with emphasis on the new genes, viral vectors and gene delivery approaches.

Osteogenic differentiation of adipose-derived stem cells and calvarial defect repair using baculovirus-mediated co-expression of BMP-2 and miR-148b

Repair of large calvarial bony defect remains a challenge for orthopedic surgeons. Since microRNAs (miRNAs) modulate the osteogenesis of osteoprogenitor cells, we aimed to engineer human adipose-derived stem cells (hASCs), a promising cell source for bone engineering, with miRNA-expressing baculovirus vectors. We constructed 4 baculoviruses each expressing 1 human miRNA (miR-26a, miR-29b, miR-148b, miR-196a) and verified that the miRNA-expressing baculovirus vectors augmented hASCs osteogenesis. Among these 4 miRNAs, miR-148b and miR-196a exerted more potent osteoinductive effects than miR-26a and miR-29b. Furthermore, we unveiled that co-transduction of hASCs with miR-148b-expressing and bone morphogenetic protein 2 (BMP-2)-expressing baculovirus vectors enhanced and prolonged BMP-2 expression, and synergistically promoted the in vitro osteogenic differentiation of hASCs. Implantation of the hASCs co-expressing BMP-2/miR-148b into critical-size (4 mm in diameter) calvarial bone defects in nude mice accelerated and potentiated the bone healing and remodeling, filling ≈94% of defect area and ≈89% of defect volume with native calvaria-like flat bone in 12 weeks, as judged from micro computed tomography, histology and immunohistochemical staining. Altogether, this study confirmed the feasibility of combining miRNA and growth factor expression for synergistic stimulation of in vitro osteogenesis and in vivo calvarial bone healing.

Graphene Oxide Triggers Toll‐Like Receptors/Autophagy Responses In Vitro and Inhibits Tumor Growth In Vivo

Graphene oxide (GO) is a nanomaterial with burgeoning bioapplications, while autophagy is implicated in cancer therapy. Although induction of autophagy by nanomaterials is reported, the underlying signaling mechanism in cancer cells and how this implicates the potential of GO in cancer therapy remain obscure. Here, it is shown that GO itself can induce the toll-like receptors (TLRs) responses and autophagy in cancer cells and confer antitumor effects in mice. GO can be phagocytosed by CT26 colon cancer cells, simultaneously triggering autophagy as well as TLR-4 and TLR-9 signaling cascades. By dissecting the crosstalk between the TLRs and autophagy pathways, it is uncovered that the GO-activated autophagy is regulated through the myeloid differentiation primary response gene 88 (MyD88)- and TNF receptor-associated factor 6 (TRAF6)-associated TLR-4/9 signaling pathways. Injection of GO alone into immunocompetent mice bearing the CT26 colon tumors not only suppresses the tumor progression but also enhances cell death, autophagy, and immune responses within the tumor bed. These data altogether implicate the potential of GO as an effective nanomaterial for autophagy induction and cancer therapy.

The use of ASCs engineered to express BMP2 or TGF-β3 within scaffold constructs to promote calvarial bone repair.

Calvarial bone healing is difficult and grafts comprising adipose-derived stem cells (ASCs) and PLGA (poly(lactic-co-glycolic acid)) scaffolds barely heal rabbit calvarial defects. Although calvarial bone forms via intramembranous ossification without cartilage templates, it was suggested that chondrocytes/cartilages promote calvarial healing, thus we hypothesized that inducing ASCs chondrogenesis and endochondral ossification involving cartilage formation can improve calvarial healing. To evaluate this hypothesis and selectively induce osteogenesis/chondrogenesis, rabbit ASCs were engineered to express the potent osteogenic (BMP2) or chondrogenic (TGF-β3) factor, seeded into either apatite-coated PLGA or gelatin sponge scaffolds, and allotransplanted into critical-size calvarial defects. Among the 4 ASCs/scaffold constructs, gelatin constructs elicited in vitro chondrogenesis, in vivo osteogenic metabolism and calvarial healing more effectively than apatite-coated PLGA, regardless of BMP2 or TGF-β3 expression. The BMP2-expressing ASCs/gelatin triggered better bone healing than TGF-β3-expressing ASCs/gelatin, filling ≈ 86% of the defect area and ≈ 61% of the volume at week 12. The healing proceeded via endochondral ossification, instead of intramembranous pathway, as evidenced by the formation of cartilage that underwent osteogenesis and hypertrophy. These data demonstrated ossification pathway switching and significantly augmented calvarial healing by the BMP2-expressing ASCs/gelatin constructs, and underscored the importance of growth factor/scaffold combinations on the healing efficacy and pathway.

Efficient gene delivery into cell lines and stem cells using baculovirus

Baculovirus is a promising vector for transducing numerous types of mammalian cells. We have developed hybrid baculovirus vectors and protocols for the efficient transduction of a variety of cell lines, primary cells and stem cells, including bone marrow–derived mesenchymal stem cells (BMSCs) and adipose-derived stem cells (ASCs). The hybrid vector enables intracellular minicircle formation and prolongs transgene expression. The advantages of this transduction protocol are that baculovirus supernatant alone needs to be added to cells growing in medium, and transduction occurs after only 4–6 h of incubation at room temperature (25 °C) with gentle shaking. The entire procedure, from virus generation to transduction, can be completed within 4 weeks. Compared with other transduction procedures, this protocol is simple and can confer efficiencies >95% for many cell types. This protocol has potential applications in tissue regeneration, as transduced cells continue to express transgenes after implantation. For example, transduction of rabbit ASCs (rASCs) with growth factor–encoding hybrid baculovirus vectors, as described as an example application in this protocol, enables robust and sustained growth factor expression, stimulates stem cell differentiation and augments tissue regeneration after implantation.

Enhanced and prolonged baculovirus-mediated expression by incorporating recombinase system and in cis elements: a comparative study

Baculovirus (BV) is a promising gene vector but mediates transient expression. To prolong the expression, we developed a binary system whereby the transgene in the substrate BV was excised by the recombinase (ΦC31o, Cre or FLPo) expressed by a second BV and recombined into smaller minicircle. The recombination efficiency was lower by ΦC31o (≈40–75%), but approached ≈90–95% by Cre and FLPo in various cell lines and stem cells [e.g. human adipose-derived stem cells (hASCs)]. Compared with FLPo, Cre exerted higher expression level and lower negative effects; thus, we incorporated additional cis-acting element [oriP/Epstein–Barr virus nuclear antigen 1 (EBNA1), scaffold/matrix attached region or human origin of replication (ori)] into the Cre-based BV system. In proliferating cells, only oriP/EBNA1 prolonged the transgene expression and maintained the episomal minicircles for 30 days without inadvertent integration, whereas BV genome was degraded in 10 days. When delivering bmp2 or vegf genes, the efficient recombination/minicircle formation prolonged and enhanced the growth factor expression in hASCs. The prolonged bone morphogenetic protein 2 expression ameliorated the osteogenesis of hASCs, a stem cell with poor osteogenesis potential. Altogether, this BV vector exploiting Cre-mediated recombination and oriP/EBNA1 conferred remarkably high recombination efficiency, which prolonged and enhanced the transgene expression in dividing and non-dividing cells, thereby broadening the applications of BV.

Graphene oxide as a chemosensitizer: Diverted autophagic flux, enhanced nuclear import, elevated necrosis and improved antitumor effects

Graphene oxide (GO) is a nanomaterial that provokes autophagy in CT26 colon cancer cells and confers antitumor effects. Here we demonstrated that both GO and the chemotherapy drug cisplatin (CDDP) induced autophagy but elicited low degrees of CT26 cell death. Strikingly, GO combined with CDDP (GO/CDDP) potentiated the CT26 cell killing via necrosis. GO/CDDP not only elicited autophagy, but induced the nuclear import of CDDP and the autophagy marker LC3. The nuclear LC3 did not co-localize with p62 or Lamp-2, neither did blocking autolysosome formation significantly hinder the nuclear import of LC3/CDDP and necrosis, indicating that autophagosome and autolysosome formation was dispensable. Conversely, suppressing phagophore formation and importin-α/β significantly alleviated the nuclear import of LC3/CDDP and necrosis. These data suggested that GO/CDDP diverted the LC3 flux in the early phase of autophagy, resulting in LC3 trafficking towards the nucleus in an importin-α/β-dependent manner, which concurred with the CDDP nuclear import and necrosis. Intratumoral injection of GO/CDDP into mice bearing CT26 colon tumors potentiated immune cell infiltration and promoted cell death, autophagy and HMGB1 release, thereby synergistically augmenting the antitumor effects. Altogether, we unveiled a mechanism concerning how nanomaterials chemosensitize cancer cells and demonstrated the potentials of GO as a chemosensitizer.

PEGylated Copper Nanowires as a Novel Photothermal Therapy Agent

Metal nanowires are promising for their applications including electrical connectors, transparent conductive electrodes and conductive additives, but the use of metal nanowires as photothermal agents to convert light to heat has yet to be reported. Here we synthesized dispersible polyethylene glycol-coated (PEGylated) copper nanowires (CuNWs) and showed for the first time that PEGylated CuNWs were able to convert near-infrared (NIR, 808 nm) light into heat at a photothermal efficiency of 12.5%. The PEGylated CuNWs exhibited good reusability and enabled rapid temperature rise to >50 °C in 6 min by NIR irradiation. The PEGylated CuNWs were flexible and intertwined around the cancer cells, which, upon NIR irradiation, allowed for direct heat transmission to cells and effectively triggered cancer cell ablation in vitro. Intratumoral injection of PEGylated CuNWs into colon tumor-bearing mice and ensuing NIR irradiation for 6 min significantly raised the local temperature to >50 °C, induced necrosis, and suppressed tumor growth. Compared with other NIR light absorbing noble metal-based nanomaterials, PEGylated CuNWs are relatively easy to synthesize in both laboratory and large scales using the low cost copper. This study demonstrated the potential of PEGylated CuNWs as a new cost-effective photothermal agent, and paved a new avenue to using CuNWs for cancer therapy.

Immune responses during healing of massive segmental femoral bone defects mediated by hybrid baculovirus-engineered ASCs.

Baculovirus holds promise for genetic modification of adipose-derived stem cells (ASCs) and bone engineering. To explore the immune responses during bone healing and the cell fate, ASCs were mock-transduced (Mock group), transduced with the baculovirus transiently expressing growth factors promoting osteogenesis (BMP2) or angiogenesis (VEGF) (S group), or transduced with hybrid baculoviruses persistently expressing BMP2/VEGF (L group). After allotransplantation into massive femoral defects in rabbits, these 3 groups triggered similar degrees of transient inflammatory response (e.g. neutrophil proliferation and immune cell infiltration into the graft site), revealing that baculovirus and transgene products did not exacerbate the inflammation. The cells in all 3 groups underwent apoptosis initially, persisted for at least 4 weeks and were eradicated thereafter. The L group prolonged the in vivo BMP2/VEGF expression (up to 4 weeks), extended the antibody responses, and slightly enhanced the cell-mediated cytotoxicity. Nonetheless, the L group led to remarkably better bone healing and remodeling than the Mock and S groups. These data confirmed that the ASCs engineered with the hybrid BV imparted prolonged expression of BMP2/VEGF which, although stimulated low levels of humoral and cell-mediated immune responses, essentially augmented the healing of massive segmental bone defects.

Baculovirus-Mediated miRNA Regulation to Suppress Hepatocellular Carcinoma Tumorigenicity and Metastasis

MicroRNA 122 (miR-122) is a tumor suppressor for hepatocellular carcinoma (HCC) but is lowly expressed in HCC cells. MiR-151 is aberrantly overexpressed in HCC cells and promotes HCC metastasis yet its roles on HCC tumorigenicity are unknown. To combat HCC tumorigenicity/metastasis, we developed Sleeping Beauty (SB)-based hybrid baculovirus (BV) vectors that expressed (i) miR-122 precursors (pre-miR-122), (ii) miR-151 sponges, or (iii) pre-miR-122 and miR-151 sponges. Transduction of aggressive HCC cells (Mahlavu) with the pre-miR-122-expressing BV tremendously enhanced miR-122 levels for >6 weeks, suppressed the levels of downstream effectors (e.g., ADAM10 and Bcl-w), proliferation, anchorage-independent growth, motility and migration/invasion in vitro. Intratumoral injection of the pre-miR-122-expressing BV attenuated the HCC growth/metastasis. The miR-151 sponges-expressing BV diminished the miR-151 levels for 6 weeks, enhanced RhoGDIA expression, suppressed RhoGTPases, as well as motility and migration/invasion of Mahlavu cells. Intratumoral injection of the miR-151 sponge-expressing BV impeded not only HCC metastasis but also cell proliferation, MMP expression and tumor growth in vivo. The BV co-expressing pre-miR-122 and miR-151 sponges also simultaneously enhanced miR-122 expression and inhibited miR-151, and conferred antitumor/anti-metastasis effects albeit lack of synergism. These data implicate the potentials of the SB-based hybrid BV for persistently modulating miRNA and suppressing HCC tumorigenicity/metastasis.