Jing Lim

Review: development of clinically relevant scaffolds for vascularised bone tissue engineering.

Clinical translation of scaffold-based bone tissue engineering (BTE) therapy still faces many challenges despite intense investigations and advancement over the years. To address these clinical barriers, it is important to analyse the current technical challenges in constructing a clinically relevant scaffold and subsequent clinical issues relating to bone repair. This review highlights the key challenges hampering widespread clinical translation of scaffold-based vascularised BTE, with a focus on the repair of large non-union defects. The main limitations of current scaffolds include the lack of sufficient vascularisation, insufficient mechanical strength as well as issues relating to the osseointegration of the bioresorbable scaffold and bone infection management. Critical insights on the current trends of scaffold technologies and future directions for advancing next-generation BTE scaffolds into the clinical realm are discussed. Considerations concerning regulatory approval and the route towards commercialisation of the scaffolds for widespread clinical utility will also be introduced.

Emerging translational research on magnetic nanoparticles for regenerative medicine.

Regenerative medicine, which replaces or regenerates human cells, tissues or organs, to restore or establish normal function, is one of the fastest-evolving interdisciplinary fields in healthcare. Over 200 regenerative medicine products, including cell-based therapies, tissue-engineered biomaterials, scaffolds and implantable devices, have been used in clinical development for diseases such as diabetes and inflammatory and immune diseases. To facilitate the translation of regenerative medicine from research to clinic, nanotechnology, especially magnetic nanoparticles have attracted extensive attention due to their unique optical, electrical, and magnetic properties and specific dimensions. In this review paper, we intend to summarize current advances, challenges, and future opportunities of magnetic nanoparticles for regenerative medicine.

An Anti-CD34 Antibody-Functionalized Clinical-Grade POSS-PCU Nanocomposite Polymer for Cardiovascular Stent Coating Applications: A Preliminary Assessment of Endothelial Progenitor Cell Capture and Hemocompatibility

In situ endothelialization of cardiovascular implants has emerged in recent years as an attractive means of targeting the persistent problems of thrombosis and intimal hyperplasia. This study aimed to investigate the efficacy of immobilizing anti-CD34 antibodies onto a POSS-PCU nanocomposite polymer surface to sequester endothelial progenitor cells (EPCs) from human blood, and to characterize the surface properties and hemocompatibility of this surface. Amine-functionalized fumed silica was used to covalently conjugate anti-CD34 to the polymer surface. Water contact angle, fluorescence microscopy, and scanning electron microscopy were used for surface characterization. Peripheral blood mononuclear cells (PBMCs) were seeded on modified and pristine POSS-PCU polymer films. After 7 days, adhered cells were immunostained for the expression of EPC and endothelial cell markers, and assessed for the formation of EPC colonies. Hemocompatibility was assessed by thromboelastography, and platelet activation and adhesion assays. The number of EPC colonies formed on anti-CD34-coated POSS-PCU surfaces was not significantly higher than that of POSS-PCU (5.0±1.0 vs. 1.7±0.6, p>0.05). However, antibody conjugation significantly improved hemocompatibility, as seen from the prolonged reaction and clotting times, decreased angle and maximum amplitude (p<0.05), as well as decreased platelet adhesion (76.8±7.8 vs. 8.4±0.7, p<0.05) and activation. Here, we demonstrate that POSS-PCU surface immobilized anti-CD34 antibodies selectively captured CD34+ cells from peripheral blood, although only a minority of these were EPCs. Nevertheless, antibody conjugation significantly improves the hemocompatibility of POSS-PCU, and should therefore continue to be explored in combination with other strategies to improve the specificity of EPC capture to promote in situ endothelialization.

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.

Cocultures of Mesenchymal Stem Cells and Endothelial Cells As Organotypic Models of Prostate Cancer Metastasis

In spite of recognized limitations in capturing species-specific responses and high costs, rodent models remain commonly used in prostate cancer metastasis research, due largely to the lack of available alternatives. We aim to develop an in vitro culture system to study prostate cancer response to a simulated bone microenvironment, which may be used to understand early events in prostate metastasis to bone or for drug screening applications. To achieve this, mesenchymal stem cells and endothelial cells were isolated and cocultured to form a vascularized bone analogue. Endothelial cells were found to exert osteopotentiating effects on mesenchymal stem cell differentiation, and reciprocal effects by the stromal cells were found to stimulate vasculogenic responses, suggesting the possible utility of this system to elicit three-way interactions between endothelial, mesenchymal, and prostate cancer cells. We further developed the use of fluorescently labeled cells which could be used to concurrently track cellular migration, proliferation, and morphometric analysis. We demonstrate the concurrent, real-time visualization of prostate cancer and endothelial cells, which may be useful for evaluation of spatiotemporal changes at a single-cell level. When prostate cancer cell proliferation on various substrates was measured, it was found that the use of coculture systems may provide a better reflection of conditions in vivo, highlighting the potential utility as a model system.

In vitro cyclic compressive loads potentiate early osteogenic events in engineered bone tissue.

Application of dynamic mechanical loads on bone and bone explants has been reported to enhance osteogenesis and mineralization. To date, published studies have incorporated a range of cyclic strains on 3D scaffolds and platforms to demonstrate the effect of mechanical loading on osteogenesis. However, most of the loading parameters used in these studies do not emulate the in vivo loading conditions. In addition, the scaffolds/platforms are not representative of the native osteoinductive environment of bone tissue and hence may not be entirely accurate to study the in vivo mechanical loading. We hypothesized that biomimicry of physiological loading will potentiate accelerated osteogenesis in bone grafts. In this study, we present a compression bioreactor system that applies cyclic compression to cellular grafts in a controlled manner. Polycaprolactone-β Tricalcium Phosphate (PCL-TCP) scaffolds seeded with Mesenchymal Stem Cells (MSC) were cyclically compressed in bioreactor for a period of 4 weeks at 1 Hz and physiological strain value of 0.22% for 4 h per day. Gene expression studies revealed increased expressions of osteogenesis-related genes (Osteonectin and COL1A1) on day 7 of cyclic loading group relative to its static controls. Cyclic compression resulted in a 3.76-fold increase in the activity of Alkaline Phosphatase (ALP) on day 14 when compared to its static group (p < 0.001). In addition, calcium deposition of cyclic loading group was found to attain saturation on day 14 (1.96 fold higher than its static scaffolds). The results suggested that cyclic, physiological compression of stem cell-seeded scaffolds generated highly mineralized bone grafts.

A Selective and Purification-Free Strategy for Labeling Adherent Cells with Inorganic Nanoparticles

Cellular labeling with inorganic nanoparticles such as magnetic iron oxide nanoparticles, quantum dots, and fluorescent silica nanoparticles is an important method for the noninvasive visualization of cells using various imaging modalities. Currently, this is mainly achieved through the incubation of cultured cells with the nanoparticles that eventually reach the intracellular compartment through specific or nonspecific internalization. This classic method is advantageous in terms of simplicity and convenience, but it suffers from issues such as difficulties in fully removing free nanoparticles (suspended in solution) and the lack of selectivity on cell types. This article reports an innovative strategy for the specific labeling of adherent cells without the concern of freely suspended nanoparticles. This method relies on a nanocomposite film that is prepared by homogeneously dispersing nanoparticles within a biodegradable polymeric film. When adherent cells are seeded on the film, they adhere, spread, and filtrate into the film through the micropores formed during the film fabrication. The pre-embedded nanoparticles are thus internalized by the cells during this infiltration process. As an example, fluorescent silica nanoparticles were homogeneously distributed within a polycaprolactone film by utilizing cryomilling and heat pressing. Upon incubation within physiological buffer, no silica nanoparticles were released from the nanocomposite film even after 20 d of incubation. However, when adherent cells (e.g., human mesenchymal stem cells) were grown on the film, they became fluorescent after 3 d, which suggests internalization of silica nanoparticles by cells. In comparison, the suspension cells (e.g., monocytes) in the medium remained nonfluorescent no matter whether there was the presence of adherent cells or not. This strategy eventually allowed the selective and concomitant labeling of mesenchymal stem cells during their harvest from bone marrow aspiration.

Human Bone Xenografts: from Preclinical Testing for Regenerative Medicine to Modeling of Diseases

Xenografting involves the transplantation of human tissue or cells into animal models and is an important tool for regenerative medicine research. Implantation of engineered human bone tissues into animal models, for example, is performed in preclinical evaluations of product safety and efficacy. With the advent of improved experimental methodologies, these models are further being exploited to interrogate molecular mechanisms and physiological interactions in vivo. In parallel to these developments, patient-derived xenograft murine models of cancer are increasingly being studied for various applications in cancer research and therapy; it follows that xenograft models in tissue engineering may be adapted for such approaches. In this review, we first discuss the development of human bone xenograft models to recapitulate physiological states in regenerative medicine. Subsequently, we discuss the use of these techniques for applications in modeling pathological states in skeletal oncology, namely, hematopoietic malignancies, bone metastatic disease, and primary bone malignancy.

Fabrication of a silver octahedral nanoparticle-containing polycaprolactone nanocomposite for antibacterial bone scaffolds

This work investigated the antimicrobial activity of Ag octahedral nanoparticle containing polycaprolactone scaffolds (Ag–PCL) that is fabricated via cryomilling. The fabricated composite scaffolds exhibited localized antibacterial activity with no adverse effects on viability and osteogenic differentiation of human fetal mesenchymal stem cells (hfMSCs). Compared to plain PCL scaffolds, the Ag–PCL scaffolds significantly reduce bacteria survival to 32.2% over a 4 hour incubation.

Surface Modification of Tissue Engineering Scaffolds

The biomaterial surface is typically engineered to elicit appropriate cellular responses. This chapter deals with commonly used methods to modify and characterise biomaterial surfaces. The first part describes surface modification technologies, broadly categorised into physical methods (comprising topographical and wettability engineering approaches) and chemical methods (comprising substantial chemical manipulation, such as plasma-based methods). In the second part, methods for the characterisation of the material are presented. These include physical assessments, where visualisation of the modified surfaces is performed, chemical assessments aimed at identifying changes in molecular compositions and, finally, biological evaluation, with a focus on cytocompatibility as an important preliminary evaluation of safety of the modified material.