Muwan Chen

Self-assembled composite matrix in a hierarchical 3-D scaffold for bone tissue engineering

It is of high clinical relevance in bone tissue engineering that scaffolds promote a high seeding efficiency of cells capable of osteogenic differentiation, such as human bone marrow-derived mesenchymal stem cells (hMSCs). We evaluated the effects of a novel polycaprolactone (PCL) scaffold on hMSC seeding efficiency, proliferation, distribution and differentiation. Porous PCL meshes prepared by fused deposition modeling (FDM) were embedded in matrix of hyaluronic acid, methylated collagen and terpolymer via polyelectrolyte complex coacervation. Scaffolds were cultured statically and dynamically in osteogenic stimulation medium for up to 28 days. Compared to naked PCL scaffolds, embedded scaffolds provided a higher cell seeding efficiency (t-test, P<0.05), a more homogeneous cell distribution and more osteogenically differentiated cells, verified by a more pronounced gene expression of the bone markers alkaline phosphatase, osteocalcin, bone sialoprotein I and bone sialoprotein II. Dynamic culture resulted in higher amounts of DNA (day 14 and day 21) and calcium (day 21 and day 28), compared to static culture. Dynamic culture and the embedding synergistically enhanced the calcium deposition of hMSC on day 21 and day 28. This in vitro study provides evidence that hybrid scaffolds made from natural and synthetic polymers improve cellular seeding efficiency, proliferation, distribution and osteogenic differentiation.

A simple method for deriving functional MSCs and applied for osteogenesis in 3D scaffolds

We describe a simple method for bone engineering using biodegradable scaffolds with mesenchymal stem cells derived from human induced-pluripotent stem cells (hiPS-MSCs). The hiPS-MSCs expressed mesenchymal markers (CD90, CD73, and CD105), possessed multipotency characterized by tri-lineages differentiation: osteogenic, adipogenic, and chondrogenic, and lost pluripotency – as seen with the loss of markers OCT3/4 and TRA-1-81 – and tumorigenicity. However, these iPS-MSCs are still positive for marker NANOG. We further explored the osteogenic potential of the hiPS-MSCs in synthetic polymer polycaprolactone (PCL) scaffolds or PCL scaffolds functionalized with natural polymer hyaluronan and ceramic TCP (PHT) both in vitro and in vivo. Our results showed that these iPS-MSCs are functionally compatible with the two 3D scaffolds tested and formed typically calcified structure in the scaffolds. Overall, our results suggest the iPS-MSCs derived by this simple method retain fully osteogenic function and provide a new solution towards personalized orthopedic therapy in the future.

Fabrication and characterization of a rapid prototyped tissue engineering scaffold with embedded multicomponent matrix for controlled drug release

Bone tissue engineering implants with sustained local drug delivery provide an opportunity for better postoperative care for bone tumor patients because these implants offer sustained drug release at the tumor site and reduce systemic side effects. A rapid prototyped macroporous polycaprolactone scaffold was embedded with a porous matrix composed of chitosan, nanoclay, and β-tricalcium phosphate by freeze-drying. This composite scaffold was evaluated on its ability to deliver an anthracycline antibiotic and to promote formation of mineralized matrix in vitro. Scanning electronic microscopy, confocal imaging, and DNA quantification confirmed that immortalized human bone marrow-derived mesenchymal stem cells (hMSC-TERT) cultured in the scaffold showed high cell viability and growth, and good cell infiltration to the pores of the scaffold. Alkaline phosphatase activity and osteocalcin staining showed that the scaffold was osteoinductive. The drug-release kinetics was investigated by loading doxorubicin into the scaffold. The scaffolds comprising nanoclay released up to 45% of the drug for up to 2 months, while the scaffold without nanoclay released 95% of the drug within 4 days. Therefore, this scaffold can fulfill the requirements for both bone tissue engineering and local sustained release of an anticancer drug in vitro. These results suggest that the scaffold can be used clinically in reconstructive surgery after bone tumor resection. Moreover, by changing the composition and amount of individual components, the scaffold can find application in other tissue engineering areas that need local sustained release of drug.

Enhanced efficacy of chemotherapy for breast cancer stem cells by simultaneous suppression of multidrug resistance and antiapoptotic cellular defense.

UNLABELLED While chemotherapy is universally recognized as a frontline treatment strategy for breast cancer, it is not always successful; among the leading causes of treatment failure is existing and/or acquired multidrug resistance. Cancer stem cells (CSCs), which constitute a minority of the cells of a tumor, are acknowledged to be responsible for increased resistance to chemo-drugs through a combination of increased expression of ATP-binding cassette transporters (ABC transporters), an increased anti-apoptotic defense, and/or the ability for extensive DNA repair like normal stem cells. Consequently, more effective therapy, especially targeted to CSCs, is urgently required. We studied the characteristics of 231-CSCs (CD44+/CD24-) sorted from human MDA-MB-231 breast cancer cells and demonstrated that 231-CSCs exhibited enhanced capacities for proliferation, migration, tumorigenesis and chemotherapy resistance. To address these multifunctional facets of CSCs, we devised a non-ionic surfactant-based vesicle (niosome) co-delivery system to simultaneously deliver siRNAs, targeted to both the ABC transporter (ABCG2) and the anti-apoptosis defense gene (BCL2), and doxorubicin (DOX) to CSCs. The rationale is to sensitize CSCs to DOX by down regulating the drug-resistance gene ABCG2 and simultaneously induce apoptosis by lowering BCL2 expression. The co-delivery system (CDS) successfully delivered siRNAs and DOX to the cytoplasm and nuclei, respectively, and resulted in a down-regulation of ABCG2- and BCL2 mRNAs in CSCs by 60% and 65%, respectively, compared to the control. A corresponding decrease in protein expression was observed using Western blotting. The IC50 of DOX in CSCs concurrently decreased significantly. Our result established CDS as a promising multi-drug delivery platform for cancer treatment. STATEMENT OF SIGNIFICANCE Cancer stem cells (CSCs) are acknowledged to be responsible for increased resistance to chemo-drugs through a combination of increased expression of ABC transporters, an increased anti-apoptotic defense, and/or the ability for extensive DNA repair like normal stem cells. Consequently, effective therapy, especially to CSCs, is urgently required. In current study, we studied the characteristics of 231-CSCs sorted from human MDA-MB-231 breast cancer cells and found that 231-CSCs possessed enhanced proliferation, migration, tumorigenesis, and DOX resistance. We employed a non-ionic surfactant-based vesicle (niosome) delivery system to simultaneously deliver siRNAs targeted to multi-drug resistance genes, and DOX to kill 231-CSCs. The CDS showed an enhanced therapeutic effect by resensitizing 231-CSCs to DOX and may constitute a promising candidate for cancer chemotherapy.

Interference in the endplate nutritional pathway causes intervertebral disc degeneration in an immature porcine model

PurposePrevious studies have shown that blocking the endplate nutritional pathway with bone cement did not result in obvious intervertebral disc degeneration (IDD) in mature animal models. However, there are very few comparable studies in immature animal models. As vertebroplasty currently is beginning to be applied in young, even biologically immature patients, it is important to investigate the effect of cement blocking at the endplate in an immature animal model.MethodsTwo lumbar intervertebral discs in eight immature pigs were either blocked by cement in both endplate pathways or stabbed with a scalpel in the annulus fibrosus (AF) as a positive control, and with a third disc remaining intact as a normal control. Magnetic resonance imaging (MRI) and histology study were performed.ResultsAfter three months, the cement-blocked discs exhibited severe IDD, with the percentage of disc-height index (DHI), nucleus pulposus (NP) area, and NP T2 value significantly lower than the normal control. These IDD changes were histologically confirmed. Post-contrast MRI showed diseased nutritional diffusion patterns in the cement-blocked discs. Moreover, the degenerative changes of the cement-blocked discs exceeded those of the injured AF positive controls.ConclusionsThe endplate nutritional pathway was interfered with and diseased after three months of bone cement intervention in an immature porcine model. Severe interference in the endplate nutritional pathway in an immature porcine model caused IDD. These findings also draw attention to the fact that interference in endplate nutritional pathways in immature or young patients may affect the vitality of adjacent discs.

A single topical dose of erythropoietin applied on a collagen carrier enhances calvarial bone healing in pigs

Background and purpose — The osteogenic potency of erythropoietin (EPO) has been documented. However, its efficacy in a large-animal model has not yet been investigated; nor has a clinically safe dosage. The purpose of this study was to overcome such limitations of previous studies and thereby pave the way for possible clinical application. Our hypothesis was that EPO increases calvarial bone healing compared to a saline control in the same subject. Methods — We used a porcine calvarial defect model. In each of 18 pigs, 6 cylindrical defects (diameter: 1 cm; height: 1 cm) were drilled, allowing 3 pairwise comparisons. Treatment consisted of either 900 IU/mL EPO or an equal volume of saline in combination with either autograft, a collagen carrier, or a polycaprolactone (PCL) scaffold. After an observation time of 5 weeks, the primary outcome (bone volume fraction (BV/TV)) was assessed with high-resolution quantitative computed tomography. Secondary outcome measures were histomorphometry and blood samples. Results — The median BV/TV ratio of the EPO-treated collagen group was 1.06 (CI: 1.02–1.11) relative to the saline-treated collagen group. Histomorphometry showed a similar median effect size, but it did not reach statistical significance. Autograft treatment had excellent healing potential and was able to completely regenerate the bone defect independently of EPO treatment. Bony ingrowth into the PCL scaffold was sparse, both with and without EPO. Neither a substantial systemic effect nor adverse events were observed. The number of blood vessels was similar in EPO-treated defects and saline-treated defects. Interpretation — Topical administration of EPO on a collagen carrier moderately increased bone healing. The dosing regime was safe, and could have possible application in the clinical setting. However, in order to increase the clinical relevance, a more potent but still clinically safe dose should be investigated.

Improvement of Distribution and Osteogenic Differentiation of Human Mesenchymal Stem Cells by Hyaluronic Acid and β-Tricalcium Phosphate-Coated Polymeric Scaffold In Vitro.

Abstract Bone tissue engineering requires a well-designed scaffold that can be biodegradable, biocompatible, and support the stem cells to osteogenic differentiation. Porous polycaprolactone (PCL) scaffold prepared by fused deposition modeling is an attractive biomaterial that has been used in clinic. However, PCL scaffolds lack biological function and osteoinductivity. In this study, we functionalized the PCL scaffolds by embedding them with a matrix of hyaluronic acid/β-tricalcium phosphate (HA/TCP). Human mesenchymal stem cells (MSCs) were cultured on scaffolds with and without coating to investigate proliferation and osteogenic differentiation. The DNA amount was significantly higher in the HA/TCP-coated scaffold on day 21. At the gene expression level, HA/TCP coating significantly increased the expression of ALP and COLI on day 4. These data correlated with the ALP activity peaking on day 7 in the HA/TCP-coated scaffold. Scanning electron microscope and histological analysis revealed that the cell matrix and calcium deposition were distributed more uniformly in the coated scaffolds compared to scaffolds without coating. In conclusion, the HA/TCP coating improved cellular proliferation, osteogenic differentiation, and uniform distribution of the cellular matrix in vitro. The HA/TCP-PCL scaffold holds great promise to accommodate human bone marrow-derived MSCs for bone reconstruction purposes, which warrants future in vivo studies.

A traditional Chinese medicine formula extracts stimulate proliferation and inhibit mineralization of human mesenchymal stem cells in vitro

AIM OF THE STUDY To investigate the effects of a traditional Chinese medicine (TCM) formula extract, named as ZD-I, on the proliferation and osteogenic differentiation of human mesenchymal stem cells (hMSCs) in vitro. MATERIALS AND METHODS When hMSCs cultivated in the basal medium with ZD-I, cell viability was assessed by MTT assay and cellular proliferation was assessed by SYBR green I assay. The effects of ZD-I on osteogenic differentiation of hMSCs were assessed by alkaline phosphatase (ALP) activity, mineralization assay and real-time RT-PCR. RESULTS ZD-I (0.78-100 microg/ml) was non-cytotoxic. The 50% inhibitory concentration (IC50) of hMSCs was 200 microg/ml. ZD-I (0.78-50 microg/ml) stimulated the proliferation of hMSCs. ZD-I did not change ALP activity of hMSCs cultivated in osteogenic medium in the early stage (4 and 7 days), but ZD-I inhibited the mineralization of hMSCs through down-regulation of several osteogenic markers (e.g. osteocalcin, bone morphogenetic protein 2 and osteopontin) in the late stage. CONCLUSIONS ZD-I stimulate cellular proliferation and decrease the bone mineral deposition of hMSCs. These results suggest ZD-I may play an important therapeutic role in osteoarthritic patients by improving proliferative capacity of hMSCs and inhibiting the mineralization of hMSCs.

Dental pulp-derived stromal cells exhibit a higher osteogenic potency than bone marrow-derived stromal cells in vitro and in a porcine critical-size bone defect model

Introduction: The osteogenic differentiation of bone marrow-derived mesenchymal stromal cells (BMSCs) was compared with that of dental pulp-derived stromal cells (DPSCs) in vitro and in a pig calvaria critical-size bone defect model. Methods: BMSCs and DPSCs were extracted from the tibia bone marrow and the molar teeth of each pig, respectively. BMSCs and DPSCs were cultured in monolayer and on a three-dimensional (3D) polycaprolactone (PCL) – hyaluronic acid – tricalcium phosphate (HT-PCL) scaffold. Population doubling (PD), alkaline phosphatase (ALP) activity, and calcium deposition were measured in monolayer. In the 3D culture ALP activity, DNA content, and calcium deposition were evaluated. Six non-penetrating critical-size defects were made in each calvarium of 14 pigs. Three paired sub-studies were conducted: (1) empty defects vs. HT-PCL scaffolds; (2) PCL scaffolds vs. HT-PCL scaffolds; and (3) autologous BMSCs on HT-PCL scaffolds vs. autologous DPSCs on HT-PCL scaffolds. The observation time was five weeks. Bone volume fractions (BV/TV) were assessed with micro-computed tomography (μCT) and histomorphometry. Results and discussion: The results from the in vitro study revealed a higher ALP activity and calcium deposition of the DPSC cultures compared with BMSC cultures. Significantly more bone was present in the HT-PCL group than in both the pure PCL scaffold group and the empty defect group in vivo. DPSCs generated more bone than BMSCs when seeded on HT-PCL. In conclusion, DPSCs exhibited a higher osteogenic potential compared with BMSCs both in vitro and in vivo, making it a potential cell source for future bone tissue engineering.

Electrostatic self-assembly of multilayer copolymeric membranes on the surface of porous tantalum implants for sustained release of doxorubicin.

Many studies in recent years have focused on surface engineering of implant materials in order to improve their biocompatibility and other performance. Porous tantalum implants have increasingly been used in implant surgeries, due to their biocompatibility, physical stability, and good mechanical strength. In this study we functionalized the porous tantalum implant for sustained drug delivery capability via electrostatic self-assembly of polyelectrolytes of hyaluronic acid, methylated collagen, and terpolymer on the surface of a porous tantalum implant. The anticancer drug doxorubicin was encapsulated into the multilayer copolymer membranes on the porous tantalum implants. Results showed the sustained released of doxorubicin from the functionalized porous tantalum implants for up to 1 month. The drug release solutions in 1 month all had inhibitory effects on the proliferation of chondrosarcoma cell line SW1353. These results suggest that this functionalized implant could be used in reconstructive surgery for the treatment of bone tumor as a local, sustained drug delivery system.