Modeling Acute Myeloid Leukemia Using StarPEG-Heparin Hydrogels.

Biomimetic semi-synthetic hydrogels formed from a combination of star-shaped poly(ethylene glycol) (starPEG) and the glycosaminoglycan, heparin, allows for the three-dimensional (3D) culture of various cells and tissues. In this chapter, we describe methods for the use of starPEG-heparin hydrogels to cultivate primary and immortalized human acute myeloid leukemia (AML) cells. The resulting 3D culture models allow for the study of AML development and response to chemotherapeutic agents.

The in situ transcriptomic landscape of breast tumour-associated and normal adjacent endothelial cells.

BACKGROUND AND AIMS: Triple Negative Breast Cancer (TNBC) is associated with increased angiogenesis, which is known to aid tumour growth and metastasis. Anti-angiogenic therapies that have been developed to target this feature have mostly generated disappointing clinical results. Further research into targeted approaches is limited by a lack of understanding of the in situ molecular profile of tumour-associated vasculature. In this study, we aimed to understand the differences in the molecular profiles of tumour endothelial cells vs normal-adjacent endothelial cells in TNBC tissues. METHOD: We have applied unbiased whole transcriptome spatial profiling of in situ gene expressions of endothelial cells localized in full-face patient TNBC tissues (n = 4) and normal-adjacent regions of the same patient breast tissues. RESULTS: Our comparative analysis revealed that 2412 genes were differentially expressed (padj < 0.05) between the tumour endothelial cells and normal-adjacent endothelial cells. Pathway enrichment showed the enrichment of gene sets related to cell-cell, cell-ECM adhesion, chromatin organization and remodeling, and protein-DNA complex subunit organization. CONCLUSION: Overall, the results revealed unique molecular profiles and signalling pathways of tumour-associated vasculature, which is a critical step towards larger cohort studies investigating potential targets for TNBC prognosis and anti-angiogenic treatments.

Microporous/Macroporous Polycaprolactone Scaffolds for Dental Applications.

This study leverages the advantages of two fabrication techniques, namely, melt-extrusion-based 3D printing and porogen leaching, to develop multiphasic scaffolds with controllable properties essential for scaffold-guided dental tissue regeneration. Polycaprolactone-salt composites are 3D-printed and salt microparticles within the scaffold struts are leached out, revealing a network of microporosity. Extensive characterization confirms that multiscale scaffolds are highly tuneable in terms of their mechanical properties, degradation kinetics, and surface morphology. It can be seen that the surface roughness of the polycaprolactone scaffolds (9.41 ± 3.01 µm) increases with porogen leaching and the use of larger porogens lead to higher roughness values, reaching 28.75 ± 7.48 µm. Multiscale scaffolds exhibit improved attachment and proliferation of 3T3 fibroblast cells as well as extracellular matrix production, compared with their single-scale counterparts (an approximate 1.5- to 2-fold increase in cellular viability and metabolic activity), suggesting that these structures could potentially lead to improved tissue regeneration due to their favourable and reproducible surface morphology. Finally, various scaffolds designed as a drug delivery device were explored by loading them with the antibiotic drug cefazolin. These studies show that by using a multiphasic scaffold design, a sustained drug release profile can be achieved. The combined results strongly support the further development of these scaffolds for dental tissue regeneration applications.

Exploring the Potential of PEG-Heparin Hydrogels to Support Long-Term Ex Vivo Culture of Patient-Derived Breast Explant Tissues.

Breast cancer is a complex, highly heterogenous, and dynamic disease and the leading cause of cancer-related death in women worldwide. Evaluation of the heterogeneity of breast cancer and its various subtypes is crucial to identify novel treatment strategies that can overcome the limitations of currently available options. Explant cultures of human mammary tissue have been known to provide important insights for the study of breast cancer structure and phenotype as they include the context of the surrounding microenvironment, allowing for the comprehensive exploration of patient heterogeneity. However, the major limitation of currently available techniques remains the short-term viability of the tissue owing to loss of structural integrity. Here, an ex vivo culture model using star-shaped poly(ethylene glycol) and maleimide-functionalized heparin (PEG-HM) hydrogels to provide structural support to the explant cultures is presented. The mechanical support allows the culture of the human mammary tissue for up to 3 weeks and prevent disintegration of the cellular structures including the epithelium and surrounding stromal tissue. Further, maintenance of epithelial phenotype and hormonal receptors is observed for up to 2 weeks of culture which makes them relevant for testing therapeutic interventions. Through this study, the importance of donor-to-donor variability and intra-patient tissue heterogeneity is reiterated.

3D Breast Tumor Models for Radiobiology Applications.

Breast cancer is a leading cause of cancer-associated death in women. The clinical management of breast cancers is normally carried out using a combination of chemotherapy, surgery and radiation therapy. The majority of research investigating breast cancer therapy until now has mainly utilized two-dimensional (2D) in vitro cultures or murine models of disease. However, there has been significant uptake of three-dimensional (3D) in vitro models by cancer researchers over the past decade, highlighting a complimentary model for studies of radiotherapy, especially in conjunction with chemotherapy. In this review, we underline the effects of radiation therapy on normal and malignant breast cells and tissues, and explore the emerging opportunities that pre-clinical 3D models offer in improving our understanding of this treatment modality.

Engineering a 3D bone marrow adipose composite tissue loading model suitable for studying mechanobiological questions.

Tissue engineering strategies are widely used to model and study the bone marrow microenvironment in healthy and pathological conditions. Yet, while bone function highly depends on mechanical stimulation, the effects of biomechanical stimuli on the bone marrow niche, specifically on bone marrow adipose tissue (BMAT) is poorly understood due to a lack of representative in vitro loading models. Here, we engineered a BMAT analog made of a GelMA (gelatin methacryloyl) hydrogel/medical-grade polycaprolactone (mPCL) scaffold composite to structurally and biologically mimic key aspects of the bone marrow microenvironment, and exploited an innovative bioreactor to study the effects of mechanical loading. Highly reproducible BMAT analogs facilitated the successful adipogenesis of human mesenchymal bone marrow stem cells. Upon long-term intermittent stimulation (1 Hz, 2 h/day, 3 days/week, 3 weeks) in the novel bioreactor, cellular proliferation and lipid accumulation were similar to unloaded controls, yet there was a significant reduction in the secretion of adipokines including leptin and adiponectin, in line with clinical evidence of reduced adipokine expression following exercise/activity. Ultimately, this innovative loading platform combined with reproducibly engineered BMAT analogs provide opportunities to study marrow physiology in greater complexity as it accounts for the dynamic mechanical microenvironment context.

Photocrosslinkable liver extracellular matrix hydrogels for the generation of 3D liver microenvironment models.

Liver extracellular matrix (ECM)-based hydrogels have gained considerable interest as biomimetic 3D cell culture environments to investigate the mechanisms of liver pathology, metabolism, and toxicity. The preparation of current liver ECM hydrogels, however, is based on time-consuming thermal gelation and limits the control of mechanical properties. In this study, we used detergent-based protocols to produce decellularized porcine liver ECM, which in turn were solubilized and functionalized with methacrylic anhydride to generate photocrosslinkable methacrylated liver ECM (LivMA) hydrogels. Firstly, we explored the efficacy of two protocols to decellularize porcine liver tissue using varying combinations of commonly used chemical agents such as Triton X-100, Sodium Dodecyl Sulphate (SDS) and Ammonium hydroxide. Then, we demonstrated successful formation of stable, reproducible LivMA hydrogels from both the protocols by photocrosslinking. The LivMA hydrogels obtained from the two decellularization protocols showed distinct mechanical properties. The compressive modulus of the hydrogels was directly dependent on the hydrogel concentration, thereby demonstrating the tuneability of mechanical properties of these hydrogels. Immortalized Human Hepatocytes cells were encapsulated in the LivMA hydrogels and cytocompatibility of the hydrogels was demonstrated after one week of culture. In summary, the LivMA hydrogel system provides a simple, photocrosslinkable platform, which can potentially be used to simulate healthy versus damaged liver for liver disease research, drug studies and cancer metastasis modelling.

In vitro engineering of a bone metastases model allows for study of the effects of antiandrogen therapies in advanced prostate cancer.

While androgen-targeted therapies are routinely used in advanced prostate cancer (PCa), their effect is poorly understood in treating bone metastatic lesions and ultimately results in the development of metastatic castrate resistant prostate cancer (mCRPC). Here, we used an all-human microtissue-engineered model of mineralized metastatic tissue combining human osteoprogenitor cells, 3D printing and prostate cancer cells, to assess the effects of the antiandrogens, bicalutamide, and enzalutamide in this microenvironment. We demonstrate that cancer/bone stroma interactions and antiandrogens drive cancer progression in a mineralized microenvironment. Probing the bone microenvironment with enzalutamide led to stronger cancer cell adaptive responses and osteomimicry than bicalutamide. Enzalutamide presented with better treatment response, in line with enzalutamide delaying time to bone-related events and enzalutamide extending survival in mCRPC. The all-human microtissue-engineered model of mineralized metastatic tissue presented here represents a substantial advance to dissect the role of the bone tumor microenvironment and responses to therapies for mCPRC.

Comparative Craniofacial Bone Regeneration Capacities of Mesenchymal Stem Cells Derived from Human Neural Crest Stem Cells and Bone Marrow.

Most craniofacial bones are derived from the ectodermal germ layer via neural crest stem cells, which are distinct from mesoderm-derived long bones. However, current craniofacial bone tissue engineering approaches do not account for this difference and utilize mesoderm-derived bone marrow mesenchymal stem cells (BM-MSCs) as a paradigm cell source. The effect of the embryonic origin (ontogeny) of an MSC population on its osteogenic differentiation potential and regenerative ability still remains unresolved. To clarify the effects of MSC ontogeny on bone regenerative ability, we directly compared the craniofacial bone regenerative abilities of an ecto-mesenchymal stem cell (eMSC) population, which is derived from human embryonic stem cells via a neural crest intermediate, with mesodermal adult BM-MSCs. eMSCs showed comparable osteogenic and chondrogenic ability to BM-MSCs in 2-D in vitro culture, but lower adipogenic ability. They exhibited greater proliferation than BM-MSCs and comparable construct mineralization in a well-established 3-D polycaprolactone-tricalcium phosphate (PCL-TCP) scaffold system in vitro. eMSC-derived 3D osteogenic constructs were maintained for longer in a proliferative osteoblast state and exhibited differential levels of genes related to fibroblast growth factor (FGF) signaling compared to BM-MSCs. Although both eMSC and BM-MSC-seeded scaffold constructs could promote bone regeneration in a rat calvarial defect model, eMSC-derived osseous constructs had significantly higher cellularity due to increased number of proliferative (Ki67+) cells than those seeded with BM-MSCs, and exhibited enhanced new bone formation in the defect area as compared to untreated controls. Overall, our study demonstrates the potential of human eMSCs for future clinical use in craniofacial regeneration applications and indicates the importance of considering MSC origin when selecting an MSC source for regenerative applications.

Convergence of 3D printed biomimetic wound dressings and adult stem cell therapy.

Biomimetically designed medical-grade polycaprolactone (mPCL) dressings are 3D-printed with pore architecture and anisotropic mechanical characteristics that favor skin wound healing with reduced scarring. Melt electrowritten mPCL dressings are seeded with human gingival tissue multipotent mesenchymal stem/stromal cells and cryopreserved using a clinically approved method. The regenerative potential of fresh or frozen cell-seeded mPCL dressing is compared in a splinted full-thickness excisional wound in a rat model over six weeks. The application of 3D-printed mPCL dressings decreased wound contracture and significantly improved skin regeneration through granulation and re-epithelialization compared to control groups. Combining 3D-printed biomimetic wound dressings and precursor cell delivery enhances physiological wound closure with reduced scar tissue formation.

Stromal fibroblasts regulate microvascular-like network architecture in a bioengineered breast tumour angiogenesis model.

The plasticity of the tumour microenvironment is a key contributor to cancer development and progression. Here, we present a bioengineered breast tumour angiogenesis model comprised of mammary derived epithelial, endothelial and fibroblast cells, to dissect the mechanisms of cancer-associated fibroblasts (CAFs) on microvascular-like network formation and epithelial spheroid morphology. Primary patient-derived mammary endothelial cells, normal breast fibroblasts (NBF, patient matched) and CAFs were cultured within three-dimensional (3D) semi-synthetic hydrogels where CAFs promoted an increase in the density and morphology of the microvascular-like network. The mammary microenvironment also increased the number of MCF-10a epithelial spheroids when compared with a non-mammary microenvironment, and a malignant mammary microenvironment resulted in further morphological differences in the epithelial spheroids. The morphological changes observed following interactions between breast CAFs and endothelial cells, highlight the plasticity of the malignant stroma in tumour vascularisation. Our in vitro bioengineered breast cancer microenvironment provides a robust model to study cell-cell and cell-matrix interactions. Statement of Significance In recent years there has been an increase in the sophistication of 3D culture models, however less attention has been paid to the cell source utilised. In this study, we describe the influence of a normal and malignant stromal microenvironment on vessel-like behaviour in a 3D model. Using a semi-synthetic hydrogel, we studied the effects of mammary-derived cancer-associated fibroblasts and normal fibroblasts on human umbilical vein endothelial cells or human mammary microvascular endothelial cells. An increase in vessel-like network and epithelial cell density was seen in a mammary versus non-mammary microenvironment. This study highlights the importance of using tissue-specific endothelial cells in cancer research and demonstrates the microenvironmental impact of fibroblasts on endothelial and epithelial growth and morphology.

Targeted camptothecin delivery via silicon nanoparticles reduces breast cancer metastasis.

In advanced breast cancer (BCa) patients, not the primary tumor, but the development of distant metastases, which occur mainly in the organ bone, and their adverse health effects are responsible for high mortality. Targeted delivery of already known drugs which displayed potency, but rather unfavorable pharmacokinetic properties, might be a promising approach to overcome the current limitations of metastatic BCa therapy. Camptothecin (CPT) is a highly cytotoxic chemotherapeutic compound, yet poorly water-soluble and non-specific. Here, CPT was loaded into porous silicon nanoparticles (pSiNP) displaying the epidermal growth factor receptor (EGFR)-targeting antibody (Ab) cetuximab to generate a soluble and targeted nanoscale delivery vehicle for cancer treatment. After confirming the cytotoxic effect of targeted CPT-loaded pSiNP in vitro on MDA-MB-231BO cells, nanoparticles were studied in a humanized BCa bone metastasis mouse model. Humanized tissue-engineered bone constructs (hTEBCs) provided a humanized microenvironment for BCa bone metastases in female NOD-scid IL2Rgnull (NSG) mice. Actively targeted CPT-loaded pSiNP led to a reduction of orthotopic primary tumor growth, increased survival rate and significant decrease in hTEBC and murine lung, liver and bone metastases. This study demonstrates that targeted delivery via pSiNP is an effective approach to employ CPT and other potent anti-cancer compounds with poor pharmacokinetic profiles in cancer therapy.

Tuning mechanical reinforcement and bioactivity of 3D printed ternary nanocomposites by interfacial peptide-polymer conjugates.

We present a study on ternary nanocomposites consisting of medical grade poly(ε-caprolactone) (mPCL) matrix, hydroxyapatite nanopowder (nHA) and compatibilized magnesium fluoride nanoparticle (cMgF2) fillers. MgF2 nanoparticles were compatibilized by following a design approach based on the material interfaces of natural bone. MgF2-specific peptide-poly(ethylene glycol) conjugates were synthesized and used as surface modifiers for MgF2 nanoparticles similarly to the non-collagenous proteins (NPC) of bone which compatibilize hydroxyapatite nanocrystallites. Different compositions of mPCL/nHA/cMgF2 composites were blended together and processed into three dimensional (3D) scaffolds using solvent-free techniques including cryomilling and melt extrusion-based additive manufacturing. The use of two different inorganic fillers in mPCL resulted in nanocomposite materials with enhanced mechanical and biological properties. In particular, cMgF2 nanoparticles were found to be the primary constitent leading to the significant improvements in the mechanical properties of these composites. The scaffolds of the ternary nanocomposites provided the best in vitro performance in terms of osteogenic differentiation and stimulated mineralization. In summary, we demonstrated that the concept of bioinspired interface engineering facilitates the development of homogeneous ternary nanocomposites with increased processability in additive biomanufacturing. Additionally, the concept leads to scaffolds exhibiting enhanced mechanical and biological properties. Overall, these multicomponent nano-interfaced building blocks add a new group of advanced functional materials with tunable mechanical properties, degradation and bioactivity.

Biomimetic fetal rotation bioreactor for engineering bone tissues-Effect of cyclic strains on upregulation of osteogenic gene expression.

Cells respond to physiological mechanical stresses especially during early fetal development. Adopting a biomimetic approach, it is necessary to develop bioreactor systems to explore the effects of physiologically relevant mechanical strains and shear stresses for functional tissue growth and development. This study introduces a multimodal bioreactor system that allows application of cyclic compressive strains on premature bone grafts that are cultured under biaxial rotation (chamber rotation about 2 axes) conditions for bone tissue engineering. The bioreactor is integrated with sensors for dissolved oxygen levels and pH that allow real-time, non-invasive monitoring of the culture parameters. Mesenchymal stem cells-seeded polycaprolactone-ß-tricalcium phosphate scaffolds were cultured in this bioreactor over 2 weeks in 4 different modes-static, cyclic compression, biaxial rotation, and multimodal (combination of cyclic compression and biaxial rotation). The multimodal culture resulted in 1.8-fold higher cellular proliferation in comparison with the static controls within the first week. Two weeks of culture in the multimodal bioreactor utilizing the combined effects of optimal fluid flow conditions and cyclic compression led to the upregulation of osteogenic genes alkaline phosphatase (3.2-fold), osteonectin (2.4-fold), osteocalcin (10-fold), and collagen type 1 α1 (2-fold) in comparison with static cultures. We report for the first time, the independent and combined effects of mechanical stimulation and biaxial rotation for bone tissue engineering using a bioreactor platform with non-invasive sensing modalities. The demonstrated results show leaning towards the futuristic vision of using a physiologically relevant bioreactor system for generation of autologous bone grafts for clinical implantation.

Review: bioreactor design towards generation of relevant engineered tissues: focus on clinical translation.

In tissue engineering and regenerative medicine, studies that utilize 3D scaffolds for generating voluminous tissues are mostly confined in the realm of in vitro research and preclinical animal model testing. Bioreactors offer an excellent platform to grow and develop 3D tissues by providing conditions that mimic their native microenvironment. Aligning the bioreactor development process with a focus on patient care will aid in the faster translation of the bioreactor technology to clinics. In this review, we discuss the various factors involved in the design of clinically relevant bioreactors in relation to their respective applications. We explore the functional relevance of tissue grafts generated by bioreactors that have been designed to provide physiologically relevant mechanical cues on the growing tissue. The review discusses the recent trends in non-invasive sensing of the bioreactor culture conditions. It provides an insight to the current technological advancements that enable in situ, non-invasive, qualitative and quantitative evaluation of the tissue grafts grown in a bioreactor system. We summarize the emerging trends in commercial bioreactor design followed by a short discussion on the aspects that hamper the 'push' of bioreactor systems into the commercial market as well as 'pull' factors for stakeholders to embrace and adopt widespread utility of bioreactors in the clinical setting. Copyright © 2017 John Wiley & Sons, Ltd.

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-ß 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. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2366-2375, 2017.

Development and Characterization of Organic Electronic Scaffolds for Bone Tissue Engineering.

Bones have been shown to exhibit piezoelectric properties, generating electrical potential upon mechanical deformation and responding to electrical stimulation with the generation of mechanical stress. Thus, the effects of electrical stimulation on bone tissue engineering have been extensively studied. However, in bone regeneration applications, only few studies have focused on the use of electroactive 3D biodegradable scaffolds at the interphase with stem cells. Here a method is described to combine the bone regeneration capabilities of 3D-printed macroporous medical grade polycaprolactone (PCL) scaffolds with the electrical and electrochemical capabilities of the conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT). PCL scaffolds have been highly effective in vivo as bone regeneration grafts, and PEDOT is a leading material in the field of organic bioelectronics, due to its stability, conformability, and biocompatibility. A protocol is reported for scaffolds functionalization with PEDOT, using vapor-phase polymerization, resulting in a conformal conducting layer. Scaffolds' porosity and mechanical stability, important for in vivo bone regeneration applications, are retained. Human fetal mesenchymal stem cells proliferation is assessed on the functionalized scaffolds, showing the cytocompatibility of the polymeric coating. Altogether, these results show the feasibility of the proposed approach to obtain electroactive scaffolds for electrical stimulation of stem cells for regenerative medicine.