Mark Seow Khoon Chong

Superior osteogenic capacity for bone tissue engineering of fetal compared with perinatal and adult mesenchymal stem cells

Mesenchymal stem cells (MSCs) from human adult bone marrow (haMSCs) represent a promising source for bone tissue engineering. However, their low frequencies and limited proliferation restrict their clinical utility. Alternative postnatal, perinatal, and fetal sources of MSCs appear to have different osteogenic capacities, but have not been systematically compared with haMSCs. We investigated the proliferative and osteogenic potential of MSCs from human fetal bone marrow (hfMSCs), human umbilical cord (hUCMSCs), and human adult adipose tissue (hATMSCs), and haMSCs, both in monolayer cultures and after loading into three‐dimensional polycaprolactone‐tricalcium‐phosphate scaffolds.Although all MSCs had comparable immunophenotypes, only hfMSCs and hUCMSCs were positive for the embryonic pluripotency markers Oct‐4 and Nanog. hfMSCs expressed the lowest HLA‐I level (55% versus 95%–99%) and the highest Stro‐1 level (51% versus 10%–27%), and had the greatest colony‐forming unit–fibroblast capacity (1.6×–2.0×; p < .01) and fastest doubling time (32 versus 54–111 hours; p < .01). hfMSCs had the greatest osteogenic capacity, as assessed by von‐Kossa staining, alkaline phosphatase activity (5.1×–12.4×; p < .01), calcium deposition (1.6×–2.7× in monolayer and 1.6×–5.0× in scaffold culture; p < .01), calcium visualized on micro‐computed tomography (3.9×17.6×; p < .01) and scanning electron microscopy, and osteogenic gene induction. Two months after implantation of cellular scaffolds in immunodeficient mice, hfMSCs resulted in the most robust mineralization (1.8×–13.3×; p < .01).The ontological and anatomical origins of MSCs have profound influences on the proliferative and osteogenic capacity of MSCs. hfMSCs had the most proliferative and osteogenic capacity of the MSC sources, as well as being the least immunogenic, suggesting they are superior candidates for bone tissue engineering. STEM CELLS 2009;27:126–137

Neo-vascularization and bone formation mediated by fetal mesenchymal stem cell tissue-engineered bone grafts in critical-size femoral defects

Tissue-engineered bone grafts (TEBG) require highly osteogenic cell sources for use in fracture repair applications. Compared to other sources of mesenchymal stem cells (MSC), human fetal MSC (hfMSC) have recently been shown to be more proliferative and osteogenic. We studied the functional performance of hfMSC-mediated TEBG in 7 mm rat femoral critical-sized bone defects (CSD). Dynamically-cultured and osteogenically-primed hfMSC seeded onto macroporous poly-epsilon-caprolactone tri-calcium phosphate scaffolds were transplanted into CSDs. After 12 weeks, hfMSC-mediated TEBG induced 2.1x more new bone formation (43.3+/-10.5 vs. 21.0+/-7.4 mm(3), p<0.05), with greater compact and woven bone, and a 9.8x increase in stiffness (3.9+/-1.7 vs. 0.4+/-0.3 mNm/degree, p<0.05) compared to acellular scaffolds, such that only animals transplanted with TEBG underwent full fracture repair of the CSD. Although hfMSC survived for <4 weeks, by 4 weeks they were associated with a 3.9x larger vasculature network in the defect area (35.2+/-11.1 vs. 6.5+/-3.6 mm(3)p<0.05), suggesting an important role for hfMSC in the promotion of neo-vasculogenesis. We speculate that hfMSC-mediated healing of the CSD by stimulating neo-vascularization through as yet undetermined mechanisms. This proof-of-principle study demonstrates the utility of primitive MSC for bone regeneration, and may be of relevance to vascularization in other areas of regenerative medicine.

Polycaprolactone-based fused deposition modeled mesh for delivery of antibacterial agents to infected wounds

Infections represent a significant source of site morbidity following tissue trauma. Scarring and tissue adhesion remain the challenging issues yet to be solved. Prolonged inflammation and morphology of the re-epithelisated layer are important considerations. We hypothesized that the solution lies not only in the biochemistry of biomaterial but also the micro-architecture of the scaffold used as the matrix for wound healing. Targeted delivery of antibiotics may provide an efficacious means of infection control through adequate release. Here, we study the use of 3-dimensional polycaprolactone-tricalcium phosphate (PCL-TCP) mesh for the delivery of gentamicin sulphate (GS) fabricated using a solvent-free method. PCL-TCP meshes incorporated with varying loads of GS were evaluated in vitro for elution profile, antimicrobial efficacy and cytotoxicity. Results showed that PCL-TCP meshes incorporated with 15 wt% GS (PT15) efficiently eliminate bacteria within 2 h and demonstrate low cytotoxicity. Subsequently, PT15 meshes were evaluated using an infected full thickness wound mice model, and observed to eliminate bacteria in the wounds effectively. Additionally, mice from the PT15 treatment group (TG) showed no observable signs of overall infection through neutrophil count by day 7 and displayed efficient wound healing (94.2% wound area reduction) by day 14. Histology also showed significantly faster healing in TG through neo-collagen deposition and wound re-epithelisation. The meshes from TG were also observed to be expelled from wounds while gauze fibers from CG were integrated into wounds during healing.

A comparison of bioreactors for culture of fetal mesenchymal stem cells for bone tissue engineering

Bioreactors provide a dynamic culture system for efficient exchange of nutrients and mechanical stimulus necessary for the generation of effective tissue engineered bone grafts (TEBG). We have shown that biaxial rotating (BXR) bioreactor-matured human fetal mesenchymal stem cell (hfMSC) mediated-TEBG can heal a rat critical sized femoral defect. However, it is not known whether optimal bioreactors exist for bone TE (BTE) applications. We systematically compared this BXR bioreactor with three most commonly used systems: Spinner Flask (SF), Perfusion and Rotating Wall Vessel (RWV) bioreactors, for their application in BTE. The BXR bioreactor achieved higher levels of cellularity and confluence (1.4-2.5x, p < 0.05) in large 785 mm(3) macroporous scaffolds not achieved in the other bioreactors operating in optimal settings. BXR bioreactor-treated scaffolds experienced earlier and more robust osteogenic differentiation on von Kossa staining, ALP induction (1.2-1.6×, p < 0.01) and calcium deposition (1.3-2.3×, p < 0.01). We developed a Micro CT quantification method which demonstrated homogenous distribution of hfMSC in BXR bioreactor-treated grafts, but not with the other three. BXR bioreactor enabled superior cellular proliferation, spatial distribution and osteogenic induction of hfMSC over other commonly used bioreactors. In addition, we developed and validated a non-invasive quantitative micro CT-based technique for analyzing neo-tissue formation and its spatial distribution within scaffolds.

Microgel Iron Oxide Nanoparticles for Tracking Human Fetal Mesenchymal Stem Cells Through Magnetic Resonance Imaging

Stem cell transplantation for regenerative medicine has made significant progress in various injury models, with the development of modalities to track stem cell fate and migration post‐transplantation being currently pursued rigorously. Magnetic resonance imaging (MRI) allows serial high‐resolution in vivo detection of transplanted stem cells labeled with iron oxide particles, but has been hampered by low labeling efficiencies. Here, we describe the use of microgel iron oxide (MGIO) particles of diameters spanning 100‐750 nm for labeling human fetal mesenchymal stem cells (hfMSCs) for MRI tracking. We found that MGIO particle uptake by hfMSCs was size dependent, with 600‐nm MGIO (M600) particles demonstrating three‐ to sixfold higher iron loading than the clinical particle ferucarbotran (33‐263 versus 9.6‐42.0 pg iron/hfMSC; p < .001). Cell labeling with either M600 particles or ferucarbotran did not affect either cellular proliferation or trilineage differentiation into osteoblasts, adipocytes, and chondrocytes, despite differences in gene expression on a genome‐wide microarray analysis. Cell tracking in a rat photothrombotic stroke model using a clinical 1.5‐T MRI scanner demonstrated the migration of labeled hfMSCs from the contralateral cortex to the stroke injury, with M600 particles achieving a five‐ to sevenfold higher sensitivity for MRI detection than ferucarbotran (p < .05). However, model‐related cellular necrosis and acute inflammation limited the survival of hfMSCs beyond 5‐12 days. The use of M600 particles allowed high detection sensitivity with low cellular toxicity to be achieved through a simple incubation protocol, and may thus be useful for cellular tracking using standard clinical MRI scanners. STEM CELLS 2009;27:1921–1931

Vasculogenic and Osteogenesis-Enhancing Potential of Human Umbilical Cord Blood Endothelial Colony-Forming Cells

Umbilical cord blood‐derived endothelial colony‐forming cells (UCB‐ECFC) show utility in neovascularization, but their contribution to osteogenesis has not been defined. Cocultures of UCB‐ECFC with human fetal‐mesenchymal stem cells (hfMSC) resulted in earlier induction of alkaline phosphatase (ALP) (Day 7 vs. 10) and increased mineralization (1.9×; p < .001) compared to hfMSC monocultures. This effect was mediated through soluble factors in ECFC‐conditioned media, leading to 1.8–2.2× higher ALP levels and a 1.4–1.5× increase in calcium deposition (p < .01) in a dose‐dependent manner. Transcriptomic and protein array studies demonstrated high basal levels of osteogenic (BMPs and TGF‐βs) and angiogenic (VEGF and angiopoietins) regulators. Comparison of defined UCB and adult peripheral blood ECFC showed higher osteogenic and angiogenic gene expression in UCB‐ECFC. Subcutaneous implantation of UCB‐ECFC with hfMSC in immunodeficient mice resulted in the formation of chimeric human vessels, with a 2.2‐fold increase in host neovascularization compared to hfMSC‐only implants (p = .001). We conclude that this study shows that UCB‐ECFC have potential in therapeutic angiogenesis and osteogenic applications in conjunction with MSC. We speculate that UCB‐ECFC play an important role in skeletal and vascular development during perinatal development but less so in later life when expression of key osteogenesis and angiogenesis genes in ECFC is lower. Stem Cells2012;30:1911–1924

The use of microgel iron oxide nanoparticles in studies of magnetic resonance relaxation and endothelial progenitor cell labelling.

In vivo tracking of stem cells after transplantation is crucial for understanding cell-fate and therapeutic efficacy. By labelling stem cells with magnetic particles, they can be tracked by Magnetic Resonance Imaging (MRI). We previously demonstrated that microgel iron oxide nanoparticle (MGIO) provide superior tracking sensitivity over commercially available particles. Here, we describe the synthesis of MGIO and report on their morphology, hydrodynamic diameters (87-766 nm), iron oxide weight content (up to 82%) and magnetization characteristics (M(s)=52.9 Am(2)/kg, M(R)=0.061 Am(2)/kg and H(c)=0.672 A/m). Their MR relaxation characteristics are comparable to those of theoretical models and represent the first such correlation between model and real particles of varying diameters. A labelling study of primary endothelial progenitor cells also confirms that MGIO is an efficient label regardless of cell type. The facile synthesis of MGIO makes it a useful tool for the studying of relaxation induced by magnetic particles and cellular tracking by MRI.

Development of cell-selective films for layered co-culturing of vascular progenitor cells

Cell-sheet assemblies are currently being studied for tissue engineering. However, tissues engineered from completely biological cell sheets lack substrate cues and possess poor mechanical strength. Recent studies demonstrate the use of synthetic bioresorbable films as scaffolds that may address these issues. Here, we describe the application of a micro-thin, biaxially-stretched polycaprolactone (muXPCL) with surface modifications for layered tissue engineering, and present the results of biphasic cell-sheet constructs using surfaces optimised for specific cell types. Polyacrylic acid (PAAc) was grafted onto muXPCL film surfaces by low-pressure plasma immobilisation. This provided a surface suitable for perivascular cells, forming the medial compartment. Subsequently, endothelial progenitor cell (EPC)-selective CD34 antibody was conjugated onto the reverse surface (intimal compartment) to select and anchor EPCs for improved adhesion and proliferation. Using the blood vessel as a model, a biphasic culture system was then setup to represent a tunica intima (endothelial cells) and tunica media (smooth muscle cells). When suitable cell types were cultured in the corresponding compartments, confluent layers of the respective populations were achieved distinctively from each other. These results demonstrate the use of muXPCL films with cell-selective modifications for layered co-cultures towards the generation of stratified tissue.

Prospective Surface Marker-Based Isolation and Expansion of Fetal Endothelial Colony-Forming Cells From Human Term Placenta

The term placenta is a highly vascularized tissue and is usually discarded upon birth. Our objective was to isolate clinically relevant quantities of fetal endothelial colony‐forming cells (ECFCs) from human term placenta and to compare them to the well‐established donor‐matched umbilical cord blood (UCB)‐derived ECFCs. A sorting strategy was devised to enrich for CD45−CD34+CD31Lo cells prior to primary plating to obtain pure placental ECFCs (PL‐ECFCs) upon culture. UCB‐ECFCs were derived using a well‐described assay. PL‐ECFCs were fetal in origin and expressed the same cell surface markers as UCB‐ECFCs. Most importantly, a single term placenta could yield as many ECFCs as 27 UCB donors. PL‐ECFCs and UCB‐ECFCs had similar in vitro and in vivo vessel forming capacities and restored mouse hind limb ischemia in similar proportions. Gene expression profiles were only minimally divergent between PL‐ECFCs and UCB‐ECFCs, probably reflecting a vascular source versus a circulating source. Finally, PL‐ECFCs and UCB‐ECFCs displayed similar hierarchies between high and low proliferative colonies. We report a robust strategy to isolate ECFCs from human term placentas based on their cell surface expression. This yielded much larger quantities of ECFCs than UCB, but the cells were comparable in immunophenotype, gene expression, and in vivo functional ability. We conclude that PL‐ECFCs have significant bio‐banking and clinical translatability potential.

Polymer Powder Processing of Cryomilled Polycaprolactone for Solvent‐Free Generation of Homogeneous Bioactive Tissue Engineering Scaffolds

Synthetic polymers used in tissue engineering require functionalization with bioactive molecules to elicit specific physiological reactions. These additives must be homogeneously dispersed in order to achieve enhanced composite mechanical performance and uniform cellular response. This work demonstrates the use of a solvent-free powder processing technique to form osteoinductive scaffolds from cryomilled polycaprolactone (PCL) and tricalcium phosphate (TCP). Cryomilling is performed to achieve micrometer-sized distribution of PCL and reduce melt viscosity, thus improving TCP distribution and improving structural integrity. A breakthrough is achieved in the successful fabrication of 70 weight percentage of TCP into a continuous film structure. Following compaction and melting, PCL/TCP composite scaffolds are found to display uniform distribution of TCP throughout the PCL matrix regardless of composition. Homogeneous spatial distribution is also achieved in fabricated 3D scaffolds. When seeded onto powder-processed PCL/TCP films, mesenchymal stem cells are found to undergo robust and uniform osteogenic differentiation, indicating the potential application of this approach to biofunctionalize scaffolds for tissue engineering applications.