Hydrogels and Bioprinting in Bone Tissue Engineering: Creating Artificial Stem-Cell Niches for In Vitro Models.

Advances in bioprinting have enabled the fabrication of complex tissue constructs with high speed and resolution. However, there remains significant structural and biological complexity within tissues that bioprinting is unable to recapitulate. Bone, for example, has a hierarchical organization ranging from the molecular to whole organ level. Current bioprinting techniques and the materials employed have imposed limits on the scale, speed, and resolution that can be achieved, rendering the technique unable to reproduce the structural hierarchies and cell-matrix interactions that are observed in bone. The shift toward biomimetic approaches in bone tissue engineering, where hydrogels provide biophysical and biochemical cues to encapsulated cells, is a promising approach to enhancing the biological function and development of tissues for in vitro modeling. A major focus in bioprinting of bone tissue for in vitro modeling is creating dynamic microenvironmental niches to support, stimulate, and direct the cellular processes for bone formation and remodeling. Hydrogels are ideal materials for imitating the extracellular matrix since they can be engineered to present various cues whilst allowing bioprinting. Here, recent advances in hydrogels and 3D bioprinting toward creating a microenvironmental niche that is conducive to tissue engineering of in vitro models of bone are reviewed.

Neutrally charged self-assembling peptide hydrogel recapitulates in vitro mechanisms of breast cancer progression.

Self-assembling peptide hydrogels (SAPH) are a popular biomaterial due to their biocompatibility with a wide range of cell types, synthetic design, structural properties that provide a more accurate 3D microenvironment, and potential for cell- and/or drug-delivery system. Mimicking solid tumors in vitro using hydrogels is one method of testing anti-cancer drug efficacy and observing cancerous cell-ECM interactions within a 3D system. In this study, a SAPH, PeptiGel®Alpha1, was used to model in vitro the 3D breast tumor microenvironment. PeptiGel®Alpha1 is composed of entangled nanofibers with consistent diameter and mechanical properties similar to breast cancer that more accurately mimic the stiffness of breast tumor tissue than Matrigel® or collagen type I. PeptiGel®Alpha1 supported the viability and growth of the breast cancer cell lines MCF-7 and MDA-MB-231 and recapitulated key features of solid tumors such as hypoxia and invasion. MCF-7 cells in the hydrogels formed large spheroids resembling acini, while MDA-MB-231 remained dispersed. When treated with tamoxifen, PeptiGel®Alpha1 acted as a barrier, providing drug penetration geometry similar to that in vivo, providing better prediction of the drug effect. Finally, it was observed that MCF-7 cells engulfed the peptide matrix after 14 days, highlighting a potential use in drug delivery. PeptiGel®Alpha1 is a suitable platform for in vitro modeling of breast cancer.

High strength and strain alginate fibers by a novel wheel spinning technique for knitting stretchable and biocompatible wound-care materials.

Alginate fibrous materials have been applied as wound dressing to enhance wound healing due to its nontoxic, biodegradable, and hemostatic nature. Conventional nonwoven fabrication tactics, however, showed weakness in inflammation, degradation stability and mechanical properties. Herein, the wet-spun alginate fibers were prepared by a novel wheel spinning technique, then knitted into wound dressing. Benefiting from optimized wet spinning parameters and the agglomeration of alginate multimers, the fibers were endowed with elevated mechanical performances and biodegradability, which allowed for the feasibility of knitting wound-care materials. Using the new wheel spinning technique, high strength alginate fibers with 173 MPa were produced with breaking strain up to 18% and toughness of 16.16 MJ*m-3. Meanwhile, alginate fibers with high breaking strain reaching 35% were produced with tensile strength of 135 MPa and toughness of 37.47 MJ*m-3. The overall mechanical performances of these alginate fibers with high breaking strain are significantly higher (up to 2 times) than those published in the literature in term of toughness. In vitro degradation evaluation revealed that this wet spun fibrous dressing had good aqueous absorbency (50%) and sustained biodegradation properties. Furthermore, the consequent cell viability study also proved that this alginate knitted fabric is biocompatible for being applied as wound dressing.

Human mesenchymal stem cells differentiate into an osteogenic lineage in presence of strontium containing bioactive glass nanoparticles.

While bioactive glass and ions released during its dissolution are known to stimulate osteoblast cells, the effect bioactive glass has on human stem cells is not clear. Here, we show that spherical monodispersed strontium containing bioactive nanoparticles (Sr-BGNPs) of composition 90.6 mol% SiO2, 5.0 mol% CaO, 4.4% mol% SrO (4.4%Sr-BGNPs) and 88.8 mol% SiO2, 1.8 mol% CaO, and 9.4 mol% SrO (9.4%Sr-BGNPs) stimulate bone marrow derived human stem cell (hMSC) differentiation down an osteogenic pathway without osteogenic supplements. The particles were synthesised using a modified StÓ§ber process and had diameters of 90 ±â€¯10 nm. Previous work on similar particles that did not contain Sr (80 mol% SiO2, 20 mol% CaO) showed stem cells did not differentiate when exposed to the particles. Here, both compositions of the Sr-BGNPs (up to concentration of 250 µg/mL) stimulated the early-, mid-, and late-stage markers of osteogenic differentiation and accelerated mineralisation in the absence of osteogenic supplements. Sr ions play a key role in osteogenic stem cell differentiation. Sr-BGNP dissolution products did not adversely affect hMSC viability and no significant differences in viability were measured between each particle composition. Confocal and transmission electron microscopy (TEM) demonstrated that monodispersed Sr-BGNPs were internalised and localised within vesicles in the cytoplasm of hMSCs. Degradation of particles inside the cells was observed, whilst maintaining effective cations (Ca and Sr) in their silica network after 24 h in culture. The uptake of Sr-BGNPs by hMSCs was reduced by inhibitors of specific routes of endocytosis, indicating that the Sr-BGNPs uptake by hMSCs was probably via mixed endocytosis mechanisms. Sr-BGNPs have potential as injectable therapeutic devices for bone regeneration or treatment of conditions such as osteoporosis, because of their ability deliver a sustained release of osteogenic inorganic cations, e.g. calcium (Ca) or and strontium (Sr), through particle degradation locally to cells. STATEMENT OF SIGNIFICANCE: Here, we show that 90 nm spherical strontium containing bioactive nanoparticles of stimulate bone marrow derived human stem cell (hMSC) differentiation down an osteogenic pathway without the use of osteogenic supplements. While bioactive glass and its dissolution products are known to promote excellent bone regeneration in vivo and to stimulate osteoblast cells to produce bone matrix in vitro, their effect on human stem cells is not clear. Previously our nanoparticles that contained only SiO2 and CaO did not provoke human bone marrow or adipose derived stem cell differentiation.

Highly degradable porous melt-derived bioactive glass foam scaffolds for bone regeneration.

A challenge in using bioactive melt-derived glass in bone regeneration is to produce scaffolds with interconnected pores while maintaining the amorphous nature of the glass and its associated bioactivity. Here we introduce a method for creating porous melt-derived bioactive glass foam scaffolds with low silica content and report in vitro and preliminary in vivo data. The gel-cast foaming process was adapted, employing temperature controlled gelation of gelatin, rather than the in situ acrylic polymerisation used previously. To form a 3D construct from melt derived glasses, particles must be fused via thermal processing, termed sintering. The original Bioglass® 45S5 composition crystallises upon sintering, altering its bioactivity, due to the temperature difference between the glass transition temperature and the crystallisation onset being small. Here, we optimised and compared scaffolds from three glass compositions, ICIE16, PSrBG and 13-93, which were selected due to their widened sintering windows. Amorphous scaffolds with modal pore interconnect diameters between 100-150µm and porosities of 75% had compressive strengths of 3.4±0.3MPa, 8.4±0.8MPa and 15.3±1.8MPa, for ICIE16, PSrBG and 13-93 respectively. These porosities and compressive strength values are within the range of cancellous bone, and greater than previously reported foamed scaffolds. Dental pulp stem cells attached to the scaffold surfaces during in vitro culture and were viable. In vivo, the scaffolds were found to regenerate bone in a rabbit model according to X-ray micro tomography imaging. STATEMENT OF SIGNIFICANCE: This manuscript describes a new method for making scaffolds from bioactive glasses using highly bioactive glass compositions. The glass compositions have lower silica content that those that have been previously made into amorphous scaffolds and they have been designed to have similar network connectivity to that of the original (and commercially used) 45S5 Bioglass. The aim was to match Bioglass' bioactivity. The scaffolds retain the amorphous nature of bioactive glass while having an open pore structure and compressive strength similar to porous bone (the original 45S5 Bioglass crystallises during sintering, which can cause reduced bioactivity or instability). The new scaffolds showed unexpectedly rapid bone regeneration in a rabbit model.

Bioinspired synthesis of hydroxyapatite nanocrystals in the presence of collagen and l-arginine: Candidates for bone regeneration.

This work aims at the bioinspired synthesis of hydroxyapatite (HAp) crystals in the presence of both collagen and l-arginine, in an effort to obtain a homogeneous hybrid material, having a bone-like nanostructure. Collagen (Col) is the most commonly utilized protein in most species of life, while L-arginine (Arg) encourages cell attachment, proliferation, and differentiation on HAp surfaces. Transmission electron microscopy, X-ray diffraction and Fourier transform-infrared spectroscopy were used to analyze surface morphology and structure of nanocrystals obtained under different synthesis conditions. It was shown that collagen and arginine content affect HAp crystallization. Collagen has an inhibition effect since HAp crystal size is reduced with the increase of collagen content. The presence of arginine is crucial as a critical content exists (Ca(2+):Arg = 1:1) under which HAp nanocrystals coexist with brushite. Under the optimum synthesis conditions (HAp/Col weight ratio 70/30 and Ca(2+):Arg molar ratio 1:1) HAp nanoplates of a uniform size (around 10 × 10 nm) were obtained. The biocompatibility of this hybrid powder was assessed using human bone marrow derived mesenchymal stem cells (MSCs). Cell response in terms of MSC attachment (scanning electron microscopy) and viability/proliferation (Alamar Blue) demonstrated a noncytotoxic effect of the new material.

Analyzing Structure and Function of Vascularization in Engineered Bone Tissue by Video-Rate Intravital Microscopy and 3D Image Processing.

Vascularization is a key challenge in tissue engineering. Three-dimensional structure and microcirculation are two fundamental parameters for evaluating vascularization. Microscopic techniques with cellular level resolution, fast continuous observation, and robust 3D postimage processing are essential for evaluation, but have not been applied previously because of technical difficulties. In this study, we report novel video-rate confocal microscopy and 3D postimage processing techniques to accomplish this goal. In an immune-deficient mouse model, vascularized bone tissue was successfully engineered using human bone marrow mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells (HUVECs) in a poly (D,L-lactide-co-glycolide) (PLGA) scaffold. Video-rate (30 FPS) intravital confocal microscopy was applied in vitro and in vivo to visualize the vascular structure in the engineered bone and the microcirculation of the blood cells. Postimage processing was applied to perform 3D image reconstruction, by analyzing microvascular networks and calculating blood cell viscosity. The 3D volume reconstructed images show that the hMSCs served as pericytes stabilizing the microvascular network formed by HUVECs. Using orthogonal imaging reconstruction and transparency adjustment, both the vessel structure and blood cells within the vessel lumen were visualized. Network length, network intersections, and intersection densities were successfully computed using our custom-developed software. Viscosity analysis of the blood cells provided functional evaluation of the microcirculation. These results show that by 8 weeks, the blood vessels in peripheral areas function quite similarly to the host vessels. However, the viscosity drops about fourfold where it is only 0.8 mm away from the host. In summary, we developed novel techniques combining intravital microscopy and 3D image processing to analyze the vascularization in engineered bone. These techniques have broad applicability for evaluating vascularization in other engineered tissues as well.

Hypoxia inducible factor-stabilizing bioactive glasses for directing mesenchymal stem cell behavior.

Oxygen tension is a known regulator of mesenchymal stem cell (MSC) plasticity, differentiation, proliferation, and recruitment to sites of injury. Materials capable of affecting the MSC oxygen-sensing pathway, independently of the environmental oxygen pressure, are therefore of immense interest to the tissue engineering (TE) and regenerative medicine community. In this study, we describe the evaluation of the effect of hypoxia inducible factor (HIF)-stabilizing bioactive glasses (BGs) on human MSCs. The dissolution products from these hypoxia-mimicking BGs stabilized HIF-1α in a concentration-dependent manner, altered cell proliferation and metabolism, and upregulated a number of genes involved in the hypoxic response (HIF1A, HIF2A, and VHL), MSC survival (SAG and BCL2), extracellular matrix remodeling (MMP1), and angiogenesis (VEGF and PDGF). These HIF-stabilizing materials can therefore be used to improve MSC survival and enhance regeneration in a number of TE strategies.

Poly(γ-glutamic acid)/silica hybrids with calcium incorporated in the silica network by use of a calcium alkoxide precursor.

Current materials used for bone regeneration are usually bioactive ceramics or glasses. Although they bond to bone, they are brittle. There is a need for new materials that can combine bioactivity with toughness and controlled biodegradation. Sol-gel hybrids have the potential to do this through their nanoscale interpenetrating networks (IPN) of inorganic and organic components. Poly(γ-glutamic acid) (γ-PGA) was introduced into the sol-gel process to produce a hybrid of γ-PGA and bioactive silica. Calcium is an important element for bone regeneration but calcium sources that are used traditionally in the sol-gel process, such as Ca salts, do not allow Ca incorporation into the silicate network during low-temperature processing. The hypothesis for this study was that using calcium methoxyethoxide (CME) as the Ca source would allow Ca incorporation into the silicate component of the hybrid at room temperature. The produced hybrids would have improved mechanical properties and controlled degradation compared with hybrids of calcium chloride (CaCl2 ), in which the Ca is not incorporated into the silicate network. Class II hybrids, with covalent bonds between the inorganic and organic species, were synthesised by using organosilane. Calcium incorporation in both the organic and inorganic IPNs of the hybrid was improved when CME was used. This was clearly observed by using FTIR and solid-state NMR spectroscopy, which showed ionic cross-linking of γ-PGA by Ca and a lower degree of condensation of the Si species compared with the hybrids made with CaCl2 as the Ca source. The ionic cross-linking of γ-PGA by Ca resulted in excellent compressive strength and reduced elastic modulus as measured by compressive testing and nanoindentation, respectively. All hybrids showed bioactivity as hydroxyapatite (HA) was formed after immersion in simulated body fluid (SBF).

Monodispersed bioactive glass submicron particles and their effect on bone marrow and adipose tissue-derived stem cells.

Spherical monodispersed bioactive particles are potential candidates for nanocomposite synthesis or as injectable particles that could be internalized by cells for the local sustained delivery of inorganic therapeutic ions (e.g., calcium or strontium). Particles are also likely to be released from porous bioactive glass and sol-gel hybrid scaffolds as they degrade; thus, it is vital to investigate their interaction with cells. Spherical monodispersed bioactive glass particles (mono-SMBG), with diameters of 215 ± 20 nm are synthesized using a modified Stöber process. Confocal and transmission electron microscopy demonstrate that mono-SMBGs are internalized by human bone marrow (MSCs) and adipose-derived stem cells (ADSCs) and located within cell vesicles and in the cytoplasm. Particle dissolution inside the cells is observed. Alamar Blue, MTT and Cyquant assays demonstrate that 50 µg mL(-1) of mono-SMBGs did not inhibit significantly MSC or ADSC metabolic activity. However, at higher concentrations (100 and 200 µg mL(-1)) small decrease in metabolic activity and total DNA is observed. Mono-SMBG did not induce ALPase activity, an early marker of osteogenic differentiation, without osteogenic supplements; however, in their presence osteogenic differentiation is achieved. Additionally, large numbers of particles are internalized by the cells but have little effect on cell behavior.

Transarterial embolization with sorafenib in animal livers: a pharmacokinetics study.

PURPOSE: To assess the safety and feasibility of the targeted delivery of the antiangiogenic drug sorafenib to the liver using transarterial chemoembolization methodology as a novel approach to hepatocellular carcinoma (HCC) therapy. MATERIALS AND METHODS: Seven healthy New Zealand white rabbits were used in the study. After placement of a catheter in the common hepatic artery, six rabbits were treated with chemoembolization of sorafenib in iodized oil (Lipiodol) (sorafenib dose 0.1 mg/kg), and one rabbit received Lipiodol only. Liquid chromatography tandem mass spectrometry was used to measure the concentration of sorafenib in the peripheral blood and liver tissue 24 hours and 72 hours after treatment. Histochemical staining of the liver sections and biochemical measurements were performed. RESULTS: The administration of sorafenib in Lipiodol emulsions by transarterial chemoembolization resulted in sorafenib concentrations of 794 ng/g ± 240 and 64 ng/g ± 15 in the liver tissue 24 hours and 72 hours after treatment. The average liver-to-serum ratios 24 hours and 72 hours after treatment were approximately 14 and 22. The histochemical staining of the liver tissue sections and aspartate aminotransferase, alanine aminotransferase, γ-glutamyltransferase and total bilirubin concentrations indicated no significant liver damage. CONCLUSIONS: Transarterial chemoembolization with sorafenib in Lipiodol is an effective methodology for the localized delivery of this drug to the liver and has possible practical implications in therapeutic interventions for the treatment of hepatocellular carcinoma.

Spherical bioactive glass particles and their interaction with human mesenchymal stem cells in vitro.

Sub-micron particles of bioactive glass (SMBGs) with composition 85 mol% SiO(2) and 15 mol% CaO were synthesised and characterised. Bioactivity was demonstrated by the formation of calcium apatite following 5 days immersion in simulated body fluid (SBF). The effect of a 24 h exposure of SMBGs (100 µg/ml, 150 µg/ml, 200 µg/ml) to human mesenchymal stem cells (hMSCs) on cell viability, metabolic activity and proliferation were determined using the LIVE/DEAD, MTT, total DNA and LDH assays after 1, 4 and 7 days of culture. None of the SMBG concentrations caused significant cytotoxicity at 1 and 4 days, but the doses of 150 and 200 µg/ml significantly decreased hMSC metabolic activity after 7 days of culture. Cell proliferation decreased as SMBG concentration increased; however none of the SMBGs tested had a significant effect on DNA quantity compared to the control. Confocal microscopy confirmed cellular uptake and localisation of the SMBGs in the hMSC cytoskeleton. Transmission electron microscopy revealed that the SMBGs localised inside the cell cytoplasm and cell endosomes. These findings are important for assessing the toxicity of sub-micron particles that may either be used as injectables for bone regeneration or generated by wear or degradation of bioactive glass scaffolds.

Engineered vascularized bone grafts.

Clinical protocols utilize bone marrow to seed synthetic and decellularized allogeneic bone grafts for enhancement of scaffold remodeling and fusion. Marrow-derived cytokines induce host neovascularization at the graft surface, but hypoxic conditions cause cell death at the core. Addition of cellular components that generate an extensive primitive plexus-like vascular network that would perfuse the entire scaffold upon anastomosis could potentially yield significantly higher-quality grafts. We used a mouse model to develop a two-stage protocol for generating vascularized bone grafts using mesenchymal stem cells (hMSCs) from human bone marrow and umbilical cord-derived endothelial cells. The endothelial cells formed tube-like structures and subsequently networks throughout the bone scaffold 4-7 days after implantation. hMSCs were essential for stable vasculature both in vitro and in vivo; however, contrary to expectations, vasculature derived from hMSCs briefly cultured in medium designed to maintain a proliferative, nondifferentiated state was more extensive and stable than that with hMSCs with a TGF-beta-induced smooth muscle cell phenotype. Anastomosis occurred by day 11, with most hMSCs associating closely with the network. Although initially immature and highly permeable, at 4 weeks the network was mature. Initiation of scaffold mineralization had also occurred by this period. Some human-derived vessels were still present at 5 months, but the majority of the graft vasculature had been functionally remodeled with host cells. In conclusion, clinically relevant progenitor sources for pericytes and endothelial cells can serve to generate highly functional microvascular networks for tissue engineered bone grafts.

Differentiation of fetal osteoblasts and formation of mineralized bone nodules by 45S5 Bioglass conditioned medium in the absence of osteogenic supplements.

Bioactive glasses bond strongly to bone in vivo and their ionic dissolution products have previously been shown to have stimulatory properties on adult and fetal osteoblasts and to induce the differentiation of embryonic stem cells towards the osteoblastic lineage in vitro. In the present study, the effect of 45S5 Bioglass conditioned medium with two different Si concentrations (15 microg/ml (BGCM/15) and 20 microg/ml (BGCM/20)) on human fetal osteoblast growth, differentiation and extracellular matrix production and mineralization was investigated. In the first instance, primary fetal osteoblasts were examined for the osteoblast phenotypic markers alkaline phosphatase (ALP), collagen type I (Col I) and OB Cadherin (Cadherin 11) (OB Cad) as well as for the mesenchymal stem cell markers CD105 and CD166. At passage 0 more than 50% of the population was positive for Col I and ALP, but at passage 2, the proportion of cells expressing ALP increased. In addition at passage 0 more than 50% of the fetal osteoblasts expressed the mesenchymal stem cell surface markers CD105 and CD166. Treatment with BGCM/15 and BGCM/20 in the absence of osteogenic supplements increased the gene expression of the bone extracellular matrix proteins alkaline phosphatase, osteonectin and bone sialoprotein as determined by quantitative real time reverse transcriptase-polymerase chain reaction (rt RT-PCR) analysis. Extracellular matrix production was also enhanced in the absence of osteogenic supplements by the 45S5 Bioglass conditioned medium as demonstrated by ALP enzymatic activity, osteocalcin and Col I protein synthesis. Furthermore, BGCM/15 and BGCM/20 significantly enhanced the formation of mineralized nodules, based on alizarin red histochemical staining, without necessitating the addition of beta-glycerophosphate, l-ascorbate-2-phosphate or dexamethasone (commonly used osteogenic supplements).

Extracellular matrix formation and mineralization on a phosphate-free porous bioactive glass scaffold using primary human osteoblast (HOB) cells.

Sol-gel derived bioactive glasses of the 70S30C (70mol% SiO2, 30mol% CaO) composition have been foamed to produce 3D bioactive scaffolds with hierarchical interconnected pore morphologies similar to trabecular bone. The aim of this study was to investigate primary human osteoblast response to porous bioactive glass scaffolds. The scaffolds supported osteoblast growth and induced differentiation, within the 3-week culture period, as depicted by enhanced ALPase enzymatic activity, without the addition of supplementary factors such as ascorbic acid, beta-glycerophosphate and dexamethasone. This is the first time this has been observed on a bioactive glass that does not contain phosphate. Deposition of extracellular matrix was also confirmed by enhanced production of the extracellular matrix protein collagen type I. SEM showed indications of mineralized bone nodule formation without the addition of growth factors. The 70S30C bioactive glass scaffolds therefore fulfil many of the criteria for an ideal scaffold for bone tissue engineering applications.