Natural killer cells activity against multiple myeloma cells is modulated by osteoblast-induced IL-6 and IL-10 production.

Background: Natural killer (NK) cells are part of the innate arm of the immune system; as such NK cells can be activated rapidly to target virus-infected cells and tumor cells without prior sensitization. The human NK-92MI cell line is among the most widely used NK cell in preclinical research studies and has also been approved for clinical applications. Previous studies have shown that osteoblasts (OSB) confer drug resistance in multiple myeloma (MM) and other cancers that metastasize to the bone marrow. Aim: We evaluated here how OSB, which are bone forming cells and a key cellular component of the bone marrow microenvironment, modulate the cytotoxic activity of NK-92MI cells against the MM.1S multiple myeloma cell line. Methods: The osteoblastic niche was recapitulated with either the osteoblastic cell line hFOB 1.19 (hFOB) or primary osteoblasts (P-OSB) derived from surgical resections. Time-lapse imaging was utilized to quantify changes in MM.1S cell viability under different conditions, including: (1) Co-culture of MM.1S with NK92MI cells, (2) triple-culture of hFOB or P-OSB with MM.1S and NK-92MI, and (3) MM.1S or NK-92MI cells primed with OSB-derived supernatant. Cytokine analysis was conducted to quantify potential secreted factors associated with the protective effects of OSB. Results: The physical presence of OSB hindered the activity of NK-92MI cells, resulting in the increased viability of MM.1S compared to co-cultures which lacked OSB. This observation was accompanied by reduced perforin and granzyme A secretion from NK-92MI cells. Contact of OSB and NK-92MI cells also induced interleukin 6 (IL-6) and interleukin 10 (IL-10) production; two cytokines which are known to impair the NK cell immunity against MM and other cancers. OSB supernatant also conferred cytoprotection to MM.1S, suggesting a dual mechanism by which OSB may modulate both NK and MM cells. Conclusions: We demonstrated here that OSB can negatively impact the activity of NK cells against MM. As NK cells and their chimeric antigen receptor-modified versions become more widely used in the clinic, our results suggest that understanding the role of OSB as potential immunoregulators of the NK cell-mediated cytotoxic response in the bone marrow tumor microenvironment may provide new opportunities for enhancing the effectiveness of this potent immunotherapeutic approach.

Hypoxic Three-Dimensional Cellular Network Construction Replicates Ex Vivo the Phenotype of Primary Human Osteocytes.

Osteocytes are deeply embedded in the mineralized matrix of bone and are nonproliferative, making them a challenge to isolate and maintain using traditional in vitro culture methods without sacrificing their inimitable phenotype. We studied the synergistic effects of two microenvironmental factors that are vital in retaining, ex vivo, the phenotype of primary human osteocytes: hypoxia and three-dimensional (3D) cellular network. To recapitulate the lacunocanalicular structure of bone tissue, we assembled and cultured primary human osteocytic cells with biphasic calcium phosphate microbeads in a microfluidic perfusion culture device. The 3D cellular network was constructed by the following: (1) the inhibited proliferation of cells entrapped by microbeads, biomimetically resembling lacunae, and (2) the connection of neighboring cells by dendrites through the mineralized, canaliculi-like interstitial spaces between the microbeads. We found that hypoxia synergistically and remarkably upregulated the mature osteocytic gene expressions of the 3D-networked cells, SOST (encoding sclerostin) and FGF23 (encoding fibroblast growth factor 23), by several orders of magnitude in comparison to those observed from two-dimensional and normoxic culture controls. Intriguingly, hypoxia facilitated the self-assembly of a nonproliferating, osteoblastic monolayer on the surface of the 3D-networked cells, replicating the osteoblastic endosteal cell layer found at the interface between native bone and bone marrow tissues. Our ability to replicate, with hypoxia, the strong expressions of these mature osteocytic markers, SOST and FGF23, is important since these (1) could not be significantly produced in vitro and (2) are new important targets for treating bone diseases. Our findings are therefore expected to facilitate ex vivo studies of human bone diseases using primary human bone cells and enable high-throughput evaluation of potential bone-targeting therapies with clinical relevance.

Ex vivo replication of phenotypic functions of osteocytes through biomimetic 3D bone tissue construction.

Osteocytes, residing as 3-dimensionally (3D) networked cells in bone, are well known to regulate bone and mineral homeostasis and have been recently implicated to interact with cancer cells to influence the progression of bone metastases. In this study, a bone tissue consisting of 3D-networked primary human osteocytes and MLO-A5 cells was constructed using: (1) the biomimetic close-packed assembly of 20-25µm microbeads with primary cells isolated from human bone samples and MLO-A5 cells and (2) subsequent perfusion culture in a microfluidic device. With this 3D tissue construction approach, we replicated ex vivo, for the first time, the mechanotransduction function of human primary osteocytes and MLO-A5 cells by correlating the effects of cyclic compression on down-regulated SOST and DKK1 expressions. Also, as an example of using our ex vivo model to evaluate therapeutic agents, we confirmed previously reported findings that parathyroid hormone (PTH) decreases SOST and increases the ratio of RANKL and OPG. In comparison to other in vitro models, our ex vivo model: (1) replicates the cell density, phenotype, and functions of primary human osteocytes and MLO-A5 cells and (2) thus provides a clinically relevant means of studying bone diseases and metastases.

Ex vivo construction of human primary 3D-networked osteocytes.

A human bone tissue model was developed by constructing ex vivo the 3D network of osteocytes via the biomimetic assembly of primary human osteoblastic cells with 20-25µm microbeads and subsequent microfluidic perfusion culture. The biomimetic assembly: (1) enabled 3D-constructed cells to form cellular network via processes with an average cell-to-cell distance of 20-25µm, and (2) inhibited cell proliferation within the interstitial confine between the microbeads while the confined cells produced extracellular matrix (ECM) to form a mechanically integrated structure. The mature osteocytic expressions of SOST and FGF23 genes became significantly higher, especially for SOST by 250 folds during 3D culture. The results validate that the bone tissue model: (1) consists of 3D cellular network of primary human osteocytes, (2) mitigates the osteoblastic differentiation and proliferation of primary osteoblast-like cells encountered in 2D culture, and (3) therefore reproduces ex vivo the phenotype of human 3D-networked osteocytes. The 3D tissue construction approach is expected to provide a clinically relevant and high-throughput means for evaluating drugs and treatments that target bone diseases with in vitro convenience.

Synchronous triple occurrence of MALT lymphoma, schwannoma, and adenocarcinoma of the stomach.

We present a case of a 56-year-old man with 3 synchronous gastric tumors. The patient presented with melena, and 3 gastric abnormalities were detected on gastroduodenoscopic examination, including a small ulcerative lesion in the gastric antrum, a submucosal mass in the gastric body, and severe erosion in the fundus. Histological examination of biopsy samples yielded respective diagnoses of gastric adenocarcinoma, gastritis, and mucosa-associated lymphoid tissue (MALT) lymphoma. The patient first received medication to eradicate any underlying Helicobacter pylori infection, which might have been a cause of the MALT lymphoma. Four weeks later, after examination of repeat biopsy samples revealed that the MALT lymphoma had resolved, the patient underwent subtotal gastrectomy. Further histological examination of resected tissue confirmed the antrum lesion as adenocarcinoma and the body lesion as schwannoma. To our knowledge, this is the first reported case of synchronous triple primary gastric adenocarcinoma, MALT lymphoma, and schwannoma.

Tissue Engineering Platforms to Replicate the Tumor Microenvironment of Multiple Myeloma.

We described here the manufacturing and implementation of two prototype perfusion culture devices designed primarily for the cultivation of difficult-to-preserve primary patient-derived multiple myeloma cells (MMC). The first device consists of an osteoblast (OSB)-derived 3D tissue scaffold constructed in a perfused microfluidic environment. The second platform is a 96-well plate-modified perfusion culture device that can be utilized to reconstruct several tissue and tumor microenvironments utilizing both primary human and murine cells. This culture device was designed and fabricated specifically to: (1) enable the preservation of primary MMC for downstream use in biological studies and chemosensitivity analyses and, (2) provide a high-throughput format that is compatible with plate readers specifically seeing that this system is built on an industry standard 96-well tissue culture plate.

Microbeads-Guided Reconstruction of 3D Osteocyte Network during Microfluidic Perfusion Culture.

Osteocytes reside as 3-dimensionally networked cells in the lacunocanalicular structure of bones, and function as the master regulators of homeostatic bone remodeling. We report here, for the first time to our best knowledge, the use of a biomimetic approach to reconstruct the 3D osteocyte network with physiological relevant microscale dimensions. In this approach, biphasic calcium phosphate microbeads were assembled with murine early osteocytes (MLO-A5) to provide an initial mechanical framework for 3D network formation and maintenance during long-term perfusion culture in a microfluidic chamber. The microbead size of 20-25 µm was used to: (1) facilitate a single cell to be placed within the interstitial space between the microbeads, (2) mitigate the proliferation of the entrapped cell due to its physical confinement in the interstitial site, and (3) control cell-to-cell distance to be 20-25 µm as observed in murine bones. The entrapped cells formed a 3D cellular network by extending and connecting their processes through openings between the microbeads within 3 days of culture. The entrapped cells produced significant mineralized extracellular matrix to fill up the interstitial spaces, resulting in the formation of a dense tissue structure during the course of 3-week culture. We found that the time-dependent osteocytic transitions of the cells exhibited trends consistent with in vivo observations, particularly with high expression of Sost gene, which is a key osteocyte-specific marker for the mechanotransduction function of osteocytes. In contrast, cells cultured in 2D well-plates did not replicate in vivo trends. These results provide an important new insight in building physiologically relevant in vitro bone tissue models.

Ex Vivo Maintenance of Primary Human Multiple Myeloma Cells through the Optimization of the Osteoblastic Niche.

We previously reported a new approach for culturing difficult-to-preserve primary patient-derived multiple myeloma cells (MMC) using an osteoblast (OSB)-derived 3D tissue scaffold constructed in a perfused microfluidic environment and a culture medium supplemented with patient plasma. In the current study, we used this biomimetic model to show, for the first time, that the long-term survival of OSB is the most critical factor in maintaining the ex vivo viability and proliferative capacity of MMC. We found that the adhesion and retention of MMC to the tissue scaffold was meditated by osteoblastic N-cadherin, as one of potential mechanisms that regulate MMC-OSB interactions. However, in the presence of MMC and patient plasma, the viability and osteogenic activity of OSB became gradually compromised, and consequently MMC could not remain viable over 3 weeks. We demonstrated that the long-term survival of both OSB and MMC could be enhanced by: (1) optimizing perfusion flow rate and patient-derived plasma composition in the culture medium and (2) replenishing OSB during culture as a practical means of prolonging MMC's viability beyond several weeks. These findings were obtained using a high-throughput well plate-based perfusion device from the perspective of optimizing the ex vivo preservation of patient-derived MM biospecimens for downstream use in biological studies and chemosensitivity analyses.

Chitosan surface enhances the mobility, cytoplasm spreading, and phagocytosis of macrophages.

A chitosan micropattern was prepared on glass by inkjet printing to visualize and compare in real-time macrophage developments on chitosan versus glass during microfluidic culture. The mobility of macrophages on chitosan was significantly higher, since the cells on glass were anchored by the development of podosomes whereas those on chitosan did not form podosomes. The phagocytosis of bacteria by macrophages was considerably more effective on chitosan because of: (1) the macrophages' higher mobility to scavenge nearby bacteria and (2) their cyotoplasm's ability to spread, re-distribute, and recover more freely to engulf the bacteria. Consequently, bacteria growth on chitosan surface was significantly reduced in the presence of macrophages in comparison to that on glass surface, as measured by surface bacteria density and effluent bacteria concentration. These findings suggest the synergistic effect of chitosan as a potential coating material on biomedical implants in promoting macrophage response upon the arrival of opportunistic bacteria.

Patient-specific 3D microfluidic tissue model for multiple myeloma.

In vitro culturing of primary multiple myeloma cells (MMC) has been a major challenge as this plasma cell malignancy depends on the bone marrow environment for its survival. Using a microfluidic platform to emulate the dynamic physiology of the bone marrow microenvironment, we report here a new approach for culturing difficult to preserve primary human MMC. The system uses a three-dimensional ossified tissue to mimic the tumor niche and recapitulate interactions between bone marrow cells and osteoblasts (OSB). To this end, the human fetal OSB cell line hFOB 1.19 was cultured in an eight-chamber microfluidic culture device to facilitate the seeding of mononuclear cells from bone marrow aspirates from three multiple myeloma patients. Optical microscopy, used for real-time monitoring of mononuclear cell interactions with the ossified tissue, confirmed that these are drawn toward the OSB layer. After 3 weeks, cocultures were characterized by flow cytometry to evaluate the amount of expansion of primary MMC (with CD138(+) and CD38(+)CD56(+) phenotypes) in this system. For each of the three patients analyzed, bone marrow mononuclear cells underwent, on an average, 2 to 5 expansions; CD38(+)CD56(+) cells underwent 1 to 3 expansions and CD138(+) cells underwent 2.5 to 4.6 expansions. This approach is expected to provide a new avenue that can facilitate: (1) testing of personalized therapeutics for multiple myeloma patients; (2) evaluation of new drugs without the need for costly animal models; and (3) studying the biology of multiple myeloma, and in particular, the mechanisms responsible for drug resistance and relapse.

Organic nanocomposite structure tailored by controlling droplet coalescence during inkjet printing.

Inkjet printing offers a low-cost, high-throughput avenue for producing functional organic materials through rapid translation of desktop discoveries to industrial roll-to-roll processes. Here, we report a simple, but effective strategy to control droplet coalescence during inkjet printing, as a major variable, to tailor the nanoscale morphology of organic composite materials produced upon evaporation of all-liquid inks. During deposition, the spacing between ink droplets was controlled to systematically vary the extent of droplet coalescence. Our results show that decreasing coalescence increased the solvent evaporation rate, supersaturation of the solutes, and nucleation density of the precipitating organic crystals. This phenomenon was utilized to tailor the average size of pentaerythritol tetranitrate (PETN) crystals dispersed in an adhesive binder matrix from ~0.2 to upwards of 100 µm. The results suggest that controlling the extent of droplet coalescence can be used as an effective means to tailor the composite morphology of printed organic materials at the nanoscale.

Microfluidic 3D bone tissue model for high-throughput evaluation of wound-healing and infection-preventing biomaterials.

We report the use of a microfluidic 3D bone tissue model, as a high-throughput means of evaluating the efficacy of biomaterials aimed at accelerating orthopaedic implant-related wound-healing while preventing bacterial infection. As an example of such biomaterials, inkjet-printed micropatterns were prepared to contain antibiotic and biphasic calcium phosphate (BCP) nanoparticles dispersed in a poly(D,L-lactic-co-glycolic) acid matrix. The micropatterns were integrated with a microfluidic device consisting of eight culture chambers. The micropatterns immediately and completely killed Staphylococcus epidermidis upon inoculation, and enhanced the calcified extracellular matrix production of osteoblasts. Without antibiotic elution, bacteria rapidly proliferated to result in an acidic microenvironment which was detrimental to osteoblasts. These results were used to demonstrate the tissue model's potential in: (i) significantly reducing the number of biomaterial samples and culture experiments required to assess in vitro efficacy for wound-healing and infection prevention and (ii) in situ monitoring of dynamic interactions of biomaterials with bacteria as wells as with tissue cells simultaneously.

Inkjet printed antibiotic- and calcium-eluting bioresorbable nanocomposite micropatterns for orthopedic implants.

Inkjet printing of antibiotic- and calcium-eluting micropatterns was explored as a novel means of preventing the formation of biofilm colonies and facilitating osteogenic cell development on orthopedic implant surfaces. The micropatterns consisted of a periodic array of ∼50 µm circular dots separated by ∼150 µm. The composition of the micropatterns was controlled by formulating inks with rifampicin (RFP) and poly(D,L-lactic-co-glycolic) acid (PLGA) dissolved in an organic solvent with ∼100 nm biphasic calcium phosphate (BCP) nanoparticles suspended in the solution. During printing RFP and PLGA co-precipitated to form a nanocomposite structure with ∼10-100 nm RFP and the BCP particles dispersed in the PLGA matrix. The rate of RFP release was strongly influenced by the RFP loading in the micropattern, particularly on the first day. The RFP-containing micropatterns effectively prevented the formation of Staphylococcus epidermidis biofilm colonies due to their ability to kill bacteria prior to forming colonies on the patterned surfaces. The BCP-containing micropatterns printed on the surface of the alloy TiAl6V4 significantly accelerated osteoblast cell differentiation, as measured by alkaline phosphatase expression and calcium deposition, without compromising cell proliferation.

RDX-based nanocomposite microparticles for significantly reduced shock sensitivity.

Cyclotrimethylenetrinitramine (RDX)-based nanocomposite microparticles were produced by a simple, yet novel spray drying method. The microparticles were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD) and high performance liquid chromatography (HPLC), which shows that they consist of small RDX crystals (∼0.1-1 µm) uniformly and discretely dispersed in a binder. The microparticles were subsequently pressed to produce dense energetic materials which exhibited a markedly lower shock sensitivity. The low sensitivity was attributed to small crystal size as well as small void size (∼250 nm). The method developed in this work may be suitable for the preparation of a wide range of insensitive explosive compositions.

Microfluidic approach to create three-dimensional tissue models for biofilm-related infection of orthopaedic implants.

With conventional in vitro culture methods, it is difficult to study complex interactions of host cells with pathogens and drugs in physiologically relevant microenvironments. To simulate orthopaedic implant-associated infection, a multi-channel microfluidic device was used to (1) observe in real-time the development of osteoblasts into three-dimensional (3D) tissue-like structures and (2) study how this development was influenced by phenotypes of Staphylococcus epidermidis. In the absence of bacteria, osteoblasts formed a confluent layer on the bottom channel surface, gradually migrated to the side and top surfaces, and formed calcified 3D nodular structures in 8 days. The delivery timing and concentration of an antibiotic were controlled to produce small colony variants, sessile biofilms, or dead cells of S. epidermidis. In the presence of the small colony variants, osteoblasts initially adhered, and spread, but were killed within 2 days. In contrast, the sessile biofilms and dead bacteria cells did not significantly interfere with the formation of tissue-like structures. The results suggest the possibility of creating in vitro tissue-biofilm-biomaterial interfaces and therefore 3D tissue models, as an entirely new method of studying biofilm-related infection of orthopaedic implants with physiological relevance.

Effects of Staphylococcus epidermidis on osteoblast cell adhesion and viability on a Ti alloy surface in a microfluidic co-culture environment.

A microfluidic device was used for real time imaging of MC3T3-E1 murine calvarial pre-osteoblasts (osteoblasts) in response to very small numbers of Staphylococcus epidermidis inoculated on the surface of a polished TiAl6V4 alloy in a serum-based medium. The Ti alloy surface was integrated to a poly(dimethylsiloxane) fluidic housing with eight 10 µl channels for high-throughput, cross-contamination-free co-culture. In the absence of S. epidermidis osteoblasts were able to adhere, spread, proliferate and remain viable on the Ti alloy surface during a 25 h culture period. With 10(2) or 10(5) colony forming units (cfu) ml(-1) S. epidermidis inoculated on the alloy surface osteoblast adhesion, spreading and proliferation were not adversely affected during the early stages of culture. However, osteoblasts became damaged by the end of culture, as S. epidermidis actively proliferated in the co-culture channels and formed small clusters on the alloy surface. These observations suggest that the small numbers of S. epidermidis did not necessarily compete with osteoblasts for the alloy surface during initial host cell development, but rapid proliferation of the bacteria might have changed the microenvironment, making it unfavorable to sustain the viability of osteoblasts. The results provide a new insight in projecting the potential utility of the microfluidic co-culture approach to developing physiologically and clinically relevant in vitro models of orthopedic implant-associated bacterial infection.

Effects of disordered hemispherical micropatterns on Staphylococcus epidermidis biofilm formation.

Surfaces which have physical patterns in the scale of bacteria cells have been shown to influence the microorganism's adhesion and biofilm formation characteristics. Layer-by-layer self-assembly was utilized to create disordered hemispherical patterns on poly(dimethylsiloxane) with a feature size of 0.5 microm, 1.0 microm and 2.0 microm. The effects of pattern size on the retention and biofilm formation of Staphylococcus epidermidis were examined as a function of culture time. The 1.0 microm pattern significantly reduced biofilm surface coverage by approximately 30% after 5 h of culture in comparison to that on an unpatterned surface while the effect of the 0.5 and 2.0 microm patterns was negligible. On the 1.0 microm surface, bacteria initially adhered on the unpatterned areas of the disordered surface and subsequently developed into biofilms by spreading across the unpatterned areas while avoiding those covered by the pattern. The results suggest that the size of surface patterns is an important factor in altering bacteria adhesion and biofilm formation characteristics.

Microfluidic devices for studying growth and detachment of Staphylococcus epidermidis biofilms.

Microfluidic devices were used to study the influences of hydrodynamics of local microenvironments on Staphylococcus epidermidis (S. epidermidis) biofilm formation and the effects of a poly(beta-1,6-N-acetyl glucosamine)-hydrolyzing enzyme (dispersin B) and/or an antibiotic (rifampicin) on the detachment of the biofilm. Elongated, monolayered biofilm morphologies were observed at high flow velocity and fluid shear locations whereas large clump-like, multilayered biofilm structures were produced at low flow velocity and fluid shear locations. Upon dispersin B treatment, most of the biofilm was detached from the microchannel surface. However, a trace amount of bacterial cells could not be removed from corner locations most likely due to the insufficient wall shear stress of the fluid at these locations. Dispersin B or rifampicin treatment was effective in delaying the dispersal behavior of bacterial cells, but could not completely remove the biofilm. Combined dynamic delivery of dispersin B and rifampicin was found to be effective for complete removal of the S. epidermidis biofilm.