In vivo delivery of VEGF RNA and protein to increase osteogenesis and intraosseous angiogenesis

Deficient bone vasculature is a key component in pathological conditions ranging from developmental skeletal abnormalities to impaired bone repair. Vascularisation is dependent upon vascular endothelial growth factor (VEGF), which drives both angiogenesis and osteogenesis. The aim of this study was to examine the efcacy of blood vessel and bone formation following transfection with VEGF RNA or delivery of recombinant human VEGF165 protein (rhVEGF165) across in vitro and in vivo model systems. To quantify blood vessels within bone, an innovative approach was developed using high-resolution X-ray computed tomography (XCT) to generate quantifable three-dimensional reconstructions. Application of rhVEGF165 enhanced osteogenesis, as evidenced by increased human osteoblast-like MG-63 cell proliferation in vitro and calvarial bone thickness following in vivo administration. In contrast, transfection with VEGF RNA triggered angiogenic efects by promoting VEGF protein secretion from MG-63VEGF165 cells in vitro, which resulted in signifcantly increased angiogenesis in the chorioallantoic (CAM) assay in ovo. Furthermore, direct transfection of bone with VEGF RNA in vivo increased intraosseous vascular branching. This study demonstrates the importance of continuous supply as opposed to a single high dose of VEGF on angiogenesis and osteogenesis and, illustrates the potential of XCT in delineating in 3D, blood vessel connectivity in bone

Genetically-programmed, mesenchymal stromal cell-laden & mechanically strong 3D bioprinted scaffolds for bone repair

Additive manufacturing processes used to create regenerative bone tissue engineered implants are not biocompatible, thereby restricting direct use with stem cells and usually require cell seeding post-fabrication. Combined delivery of stem cells with the controlled release of osteogenic factors, within a mechanically-strong biomaterial combined during manufacturing would replace injectable defect fillers (cements) and allow personalized implants to be rapidly prototyped by 3D bioprinting.Through the use of direct genetic programming via the sustained release of an exogenously delivered transcription factor RUNX2 (delivered as recombinant GET-RUNX2 protein) encapsulated in PLGA microparticles (MPs), we demonstrate that human mesenchymal stromal (stem) cells (hMSCs) can be directly fabricated into a thermo-sintered 3D bioprintable material and achieve effective osteogenic differentiation. Importantly we observed osteogenic programming of gene expression by released GET-RUNX2 (8.2-, 3.3- and 3.9-fold increases in OSX, RUNX2 and OPN expression, respectively) and calcification (von Kossa staining) in our scaffolds. The developed biodegradable PLGA/PEG paste formulation augments high-density bone development in a defect model (~2.4-fold increase in high density bone volume) and can be used to rapidly prototype clinically-sized hMSC-laden implants within minutes using mild, cytocompatible extrusion bioprinting.The ability to create mechanically strong 'cancellous bone-like’ printable implants for tissue repair that contain stem cells and controlled-release of programming factors is innovative, and will facilitate the development of novel localized delivery approaches to direct cellular behaviour for many regenerative medicine applications including those for personalized bone repair

Biomimetic generation of the strongest known biomaterial found in limpet tooth

The biomaterial with the highest known tensile strength is a unique composite of chitin and goethite (α-FeO(OH)) present in teeth from the Common Limpet (Patella vulgata). A biomimetic based on limpet tooth, with corresponding high-performance mechanical properties is highly desirable. Here we report on the replication of limpet tooth developmental processes ex vivo, where isolated limpet tissue and cells in culture generate new biomimetic structures. Transcriptomic analysis of each developmental stage of the radula, the organ from which limpet teeth originate, identifies sequential changes in expression of genes related to chitin and iron processing. We quantify iron and chitin metabolic processes in the radula and grow isolated radula cells in vitro. Bioinspired material can be developed with electrospun chitin mineralised by conditioned media from cultured radula cells. Our results inform molecular processes behind the generation of limpet tooth and establish a platform for development of a novel biomimetic with comparable properties

The hypoxic cancer secretome induces pre-metastatic bone lesions through lysyl oxidase

Tumour metastasis is a complex process involving reciprocal interplay between cancer cells and host stroma at both primary and secondary sites, and is strongly influenced by microenvironmental factors such as hypoxia. Tumour-secreted proteins play a crucial role in these interactions and present strategic therapeutic potential. Metastasis of breast cancer to the bone affects approximately 85% of patients with advanced disease and renders them largely untreatable. Specifically, osteolytic bone lesions, where bone is destroyed, lead to debilitating skeletal complications and increased patient morbidity and mortality. The molecular interactions governing the early events of osteolytic lesion formation are currently unclear. Here we show hypoxia to be specifically associated with bone relapse in patients with oestrogen-receptor negative breast cancer. Global quantitative analysis of the hypoxic secretome identified lysyl oxidase (LOX) as significantly associated with bone-tropism and relapse. High expression of LOX in primary breast tumours or systemic delivery of LOX leads to osteolytic lesion formation whereas silencing or inhibition of LOX activity abrogates tumour-driven osteolytic lesion formation. We identify LOX as a novel regulator of NFATc1-driven osteoclastogenesis,independent of RANK ligand, which disrupts normal bone homeostasisleading to the formation of focal pre-metastatic lesions. We show that these lesions subsequently provide a platform for circulating tumour cells to colonize and form bone metastases. Our study identifies a novel mechanism of regulation of bone homeostasis and metastasis, opening up opportunities for novel therapeutic intervention with important clinical implications

Doxycycline reduces the number of macrophages surrounding mammary tumors in PyMT-MacLow mice.

<p>(<b>A</b>) Sections of mammary tissue were labelled for F4/80 (DAB brown cells) and counterstained with haematoxylin; a representative image at the early carcinoma stage of tumor development is shown for each genotype and treatment. (<b>B</b>) Images captured from slides scanned on an Aperio slide scanner were used to quantify the number of macrophages within (intratumoral) and on the perimeter of mammary tumors (peritumoral). The data was then grouped according to genotype and treatment group and the mean value +/- SD shown. Each individual data point represent the mean values for an individual tumor. Tumor samples were obtained from the following numbers of animals per treatment group the number of tumors analysed is indicated in brackets: PyMT UT = 5(27), PyMT Doxy = 5(33), PyMT-MacLow UT = 7(31), PyMT-MacLow Doxy = 7(39). Significance is from the SPSS nested analysis comparing data from doxycycline treated versus control animals for each tumor and genotype. NS = not significant, **P<0.01. Scalebar = 100 μm. (C) The percentage of animals that had any tumors of hyperplasia and/or Adenoma/mammary intraepithelial neoplasia stages (Adenoma/MIN) was calculated for each genotype and treatment group. A Chi-square test was used to calculate statistical significance.</p

CD68+ and F4/80+ macrophages are analogous populations in PyMT tumors.

<p>Tumors from PyMT mice were enzymatically digested and made into a single cell suspension. Cells were stained with antibodies against the surface markers CD45, CD11b and F4/80. Cells were then fixed, permeabilized and stained with anti-CD68. Fluorescence was measured by flow cytometry and the data was analyzed using Flowjo software. Dot plots shown were generated from CD45⁺ cells. CD68 expression was determined after gating on tumor-associated macrophages (CD45⁺CD11b⁺F4/80⁺), CD11b⁺F4/80^—cells and CD11b^—F4/80^—cells. Representative dot plots are shown from one of four mice analysed.</p

The proliferative capacity of Adenoma/MIN tumors is reduced in macrophage deficient mice.

<p>Mammary sections were labelled with an anti-Ki67 antibody as a marker of proliferation and counterstained with haematoxylin. (<b>A</b>) Images were captured from slides scanned on an Aperio slide scanner and split according to tumor grade, a representative image of an Adeno/MIN tumor (A/M) is shown for each genotype and treatment group. (<b>B</b>) The amount of Ki67 labelling in individual tumor areas (cells stained dark brown with DAB) was quantified and expressed as positivity using the Aperio positive pixel algorithm. Each data point on the graph represents the mean positivity for an individual tumor and the number of animals these tumors were taken from is shown below the x axis on each graph. The mean value ± SD was plotted and a nested analysis carried out in SPSS to compare data from doxycycline treated versus control animals from each tumor grade and genotype. ***P<0.001. Scalebar = 100 μm.</p