Neutrophils contribute to fracture healing by synthesizing fibronectin+ extracellular matrix rapidly after injury.

The role of inflammatory cells in bone regeneration remains unclear. We hypothesize that leukocytes contribute to fracture healing by rapidly synthesizing an "emergency extracellular matrix (ECM)" before stromal cells infiltrate the fracture hematoma (FH) and synthesize the eventual collagenous bone tissue. 53 human FHs were isolated at different time points after injury, ranging from day 0 until day 23 after trauma and stained using (immuno)histochemistry. FHs isolated within 48 h after injury contained fibronectin(+) ECM, which increased over time. Neutrophils within the early FHs stained positive for cellular fibronectin and neutrophil derived particles were visible within the fibronectin(+) ECM. Stromal cells appeared at day 5 after injury or later and collagen type I birefringent fibrils could be identified during the second week after injury. Our study suggests that neutrophils contribute to bone regeneration by synthesizing an "emergency ECM" before stromal cells infiltrate the FH and synthesize the eventual bone tissue.

Suppression of the immune system as a critical step for bone formation from allogeneic osteoprogenitors implanted in rats.

The surface marker profile of mesenchymal stromal cells (MSCs) suggests that they can escape detection by the immune system of an allogeneic host. This could be an optimal strategy for bone regeneration applications, where off-the-shelf cells could be implanted to heal bone defects. However, it is unknown how pre-differentiation of MSCs to an osteogenic lineage, a means of improving bone formation, affects their immunogenicity. Using immunohistological techniques in a rat ectopic implantation model, we demonstrate that allogeneic osteoprogenitors mount a T cell- and B cell-mediated immune response resulting in an absence of in vivo bone formation. Suppression of the host immune response with daily administration of an immunosuppressant, FK506, is effective in preventing the immune attack on the allogeneic osteoprogenitors. In the immunosuppressed environment, the allogeneic osteoprogenitors are capable of generating bone in amounts similar to those of syngeneic cells. However, using osteoprogenitors from one of the allogeneic donors led to newly deposited bone that was attacked by the host immune system, despite the continued administration of the immunosuppressant. This suggests that, although using an immunosuppressant can potentially suppress the immune attack on the allogeneic cells, optimizing the dose of the immunosuppressant may be crucial to ensure bone formation within the allogeneic environment. Overall, allografts comprising osteoprogenitors derived from allogeneic MSCs have the potential to be used in bone regeneration applications.

Non-viral gene therapy for bone tissue engineering.

The possibilities of using gene therapy for bone regeneration have been extensively investigated. Improvements in the design of new transfection agents, combining vectors and delivery/release systems to diminish cytotoxicity and increase transfection efficiencies have led to several successful in vitro, ex vivo and in vivo strategies. These include growth factor or short interfering ribonucleic acid (siRNA) delivery, or even enzyme replacement therapies, and have led to increased osteogenic differentiation and bone formation in vivo. These results provide optimism to consider use in humans with some of these gene-delivery strategies in the near future.

The Added Value of the "Co" in Co-Culture Systems in Research on Osteoarthritis Pathology and Treatment Development.

Osteoarthritis (OA) is a highly prevalent disease and a major health burden. Its development and progression are influenced by factors such as age, obesity or joint overuse. As a whole organ disease OA affects not only cartilage, bone and synovium but also ligaments, fatty or nervous tissue surrounding the joint. These joint tissues interact with each other and understanding this interaction is important in developing novel treatments. To incorporate and study these interactions in OA research, several co-culture models have evolved. They combine two or more cell types or tissues and investigate the influence of amongst others inflammatory or degenerative stimuli seen in OA. This review focuses on co-cultures and the differential processes occurring in a given tissue or cell as a consequence of being combined with another joint cell type or tissue, and/or the extent to which a co-culture mimics the in vivo processes. Most co-culture models depart from synovial lining and cartilage culture, but also fat pad and bone have been included. Not all of the models appear to reflect the postulated in vivo OA pathophysiology, although some of the discrepancies may indicate current assumptions on this process are not entirely valid. Systematic analysis of the mutual influence the separate compartments in a given model exert on each other and validation against in vivo or ex vivo observation is still largely lacking and would increase their added value as in vitro OA models.

Use of Therapeutic Pathogen Recognition Receptor Ligands for Osteo-Immunomodulation.

Therapeutic pathogen recognition receptor (PRR) ligands are reaching clinical practice following their ability to skew the immune response in a specific direction. We investigated the effects of various therapeutic PRR ligands on bone cell differentiation and inflammation. Following stimulation, alkaline phosphatase (ALP) activity (Day 10), osteocalcin, osteonectin expression (Day 14), and calcium deposition (Day 21) were quantified in bone marrow-derived human mesenchymal stem cells (hMSCs). The osteoclastogenic response was determined by measuring tartrate-resistant acid phosphate (TRAP) activity in human monocytes. TNF-α, IL-6, IL-8, and IL-10 expressions were measured by enzyme-linked immunosorbent assay as an indicator of the ligands' inflammatory properties. We found that nucleic acid-based ligands Poly(I:C) and CpG ODN C increased early ALP activity in hMSCs by 4-fold without affecting osteoclast formation. These ligands did not enhance expression of the other, late osteogenic markers. MPLA, Curdlan, and Pam3CSK4 did not affect osteogenic differentiation, but inhibited TRAP activity in monocytes, which was associated with increased expression of all measured cytokines. Nucleic acid-based ligands are identified as the most promising osteo-immunomodulators, as they favor early osteogenic differentiation without inducing an exaggerated immune-cell mediated response or interfering in osteoclastogenesis and thus can be potentially harnessed for multifunctional coatings for bone biomaterials.

Flavonoids influence monocytic GTPase activity and are protective in experimental allergic encephalitis.

In the chronic disabling disease multiple sclerosis (MS), migration of monocytes across the blood-brain barrier is a crucial step in the formation of new lesions in the central nervous system (CNS). Infiltrating monocyte-derived macrophages secrete inflammatory mediators such as oxygen radicals, which contribute to axonal demyelination and damage, resulting in neurological deficits. Flavonoids are compounds occurring naturally in food, which scavenge oxygen radicals and have antiinflammatory properties. To investigate whether they might suppress clinical symptoms in MS, we treated rats sensitized for acute and chronic experimental allergic encephalomyelitis, an experimental model of MS, with flavonoids. We demonstrated that the flavonoid luteolin substantially suppressed clinical symptoms and prevented relapse when administered either before or after disease onset. Luteolin treatment resulted in reduced inflammation and axonal damage in the CNS by preventing monocyte migration across the brain endothelium. Luteolin influenced migration by modulating the activity of Rho GTPases, signal transducers involved in transendothelial migration. Oral administration of luteolin also significantly reduced clinical symptoms.

Evaluation of photocrosslinked Lutrol hydrogel for tissue printing applications.

Application of hydrogels in tissue engineering and innovative strategies such as organ printing, which is based on layered 3D deposition of cell-laden hydrogels, requires design of novel hydrogel matrices. Hydrogel demands for 3D printing include: 1) preservation of the printed shape after the deposition; 2) maintaining cell viability and cell function and 3) easy handling of the printed construct. In this study we analyze the applicability of a novel, photosensitive hydrogel (Lutrol) for printing of 3D structured bone grafts. We benefit from the fast temperature-responsive gelation ability of thermosensitive Lutrol-F127, ensuring organized 3D extrusion, and the additional stability provided by covalent photocrosslinking allows handling of the printed scaffolds. We studied the cytotoxicity of the hydrogel and osteogenic differentiation of embedded osteogenic progenitor cells. After photopolymerization of the modified Lutrol hydrogel, cells remain viable for up to three weeks and retain the ability to differentiate. Encapsulation of cells does not compromise the mechanical properties of the formed gels and multilayered porous Lutrol structures were successfully printed.

BMP-2 gene delivery in cell-loaded and cell-free constructs for bone regeneration.

To induce osteogenicity in bone graft substitutes, plasmid-based expression of BMP-2 (pBMP-2) has been successfully applied in gene activated matrices based on alginate polymer constructs. Here, we investigated whether cell seeding is necessary for non-viral BMP-2 gene expression in vivo. Furthermore, to gain insight in the role of BMP-producing cells, we compared inclusion of bone progenitor cells with non-osteogenic target cells in gene delivery constructs. Plasmid DNA encoding GFP (pGFP) was used to trace transfection of host tissue cells and seeded cells in a rat model. Transgene expression was followed in both cell-free alginate-ceramic constructs as well as constructs seeded with syngeneic fibroblasts or multipotent mesenchymal stromal cells (MSCs). Titration of pGFP revealed that the highest pGFP dose resulted in frequent presence of positive host cells in the constructs. Both cell-loaded groups were associated with transgene expression, most effectively in the MSC-loaded constructs. Subsequently, we investigated effectiveness of cell-free and cell-loaded alginate-ceramic constructs with pBMP-2 to induce bone formation. Local BMP-2 production was found in all groups containing BMP-2 plasmid DNA, and was most pronounced in the groups with MSCs transfected with high concentration pBMP-2. Bone formation was only apparent in the recombinant protein BMP-2 group. In conclusion, we show that non-viral gene delivery of BMP-2 is a potentially effective way to induce transgene expression in vivo, both in cell-seeded as well as cell-free conditions. However, alginate-based gene delivery of BMP-2 to host cells or seeded cells did not result in protein levels adequate for bone formation in this setting, calling for more reliable scaffold compatible transfection methods.

A Human Hematopoietic Niche Model Supporting Hematopoietic Stem and Progenitor Cells In Vitro.

Niches in the bone marrow regulate hematopoietic stem and progenitor cell (HSPC) fate and behavior through cell-cell interactions and soluble factor secretion. The niche-HSPC crosstalk is a very complex process not completely elucidated yet. To aid further investigation of this crosstalk, a functional in vitro 3D model that closely represents the main supportive compartments of the bone marrow is developed. Different combinations of human stromal cells and hydrogels are tested for their potential to maintain CD34+ HSPCs. Cell viability, clonogenic hematopoietic potential, and surface marker expression are assessed over time. Optimal HSPC support is obtained in presence of adipogenic and osteogenic cells, together with progenitor derived endothelial cells. When cultured in a bioactive hydrogel, the supportive cells self-assemble into a hypoxic stromal network, stimulating CD34+ CD38+ cell formation, while maintaining the pool of CD34+ 38- HSPCs. HSPC clusters colocalize with the stromal networks, in close proximity to sinusoidal clusters of CD31+ endothelial cells. Importantly, the primary in vitro niche model supports HSPCs with no cytokine addition. Overall, the engineered primary 3D bone marrow environment provides an easy and reliable model to further investigate interactions between HSPCs and their endosteal and perivascular niches, in the context of normal hematopoiesis or blood-related diseases.

The Osteoinductive Effect of Controlled Bone Morphogenic Protein 2 Release Is Location Dependent.

IMPACT STATEMENT: The main challenge in bone morphogenic protein 2 (BMP-2)-based application lies in finding strategies to prolong its biologic activity as it has a short biological half-life. The present study uses a phosphate-modified oligo[(polyethylene glycol) fumarate] hydrogel that can be tuned to achieve differential release profiles of biologically active BMP-2 release. We demonstrate that this platform outperforms Infuse®, currently used in the clinic and that the osteoinductive effect of BMP-2 is location dependent. Altogether, this study stresses the importance of evaluating efficacy of bone tissue engineering strategies at an orthotopic location rather than subcutaneously. Even more so, it emphasizes the role of biomaterials as a scaffold to achieve proper bone tissue engineering.

Photopolymerized thermosensitive hydrogels: synthesis, degradation, and cytocompatibility.

In situ forming hydrogels based on thermosensitive polymers have attractive properties for tissue engineering. However, the physical interactions in these hydrogels are not strong enough to yield gels with sufficient stability for many of the proposed applications. In this study, additional covalent cross-links were introduced by photopolymerization to improve the mechanical properties and the stability of thermosensitive hydrogels. Methacrylate groups were coupled to the side chains of triblock copolymers (ABA) with thermosensitive poly( N-(2-hydroxypropyl) methacrylamide lactate) A blocks and a hydrophilic poly(ethylene glycol) B block. These polymers exhibit lower critical solution temperature (LCST) behavior in aqueous solution and the cloud point decreased with increasing amounts of methacrylate groups. These methacrylate groups were photopolymerized above the LCST to render covalent cross-links within the hydrophobic domains. The mechanical properties of photopolymerized hydrogels were substantially improved and their stability was prolonged significantly compared to nonphotopolymerized hydrogels. Whereas non-UV-cured gels disintegrated within 2 days at physiological pH and temperature, the photopolymerized gels degraded in 10 to 25 days depending on the degree of cross-linking. To assess biocompatibility, goat mesenchymal stem cells were seeded on the hydrogel surface or encapsulated within the gel and they remained viable as demonstrated by a LIVE/DEAD cell viability/cytotoxicity assay. Expression of alkaline phosphatase and production of collagen I demonstrated the functionality of the mesenchymal stem cells and their ability to differentiate upon encapsulation. Due to the improved mechanical properties, stability, and adequate cytocompatibility, the photopolymerized thermosensitive hydrogels can be regarded as highly potential materials for applications in tissue engineering.

Signal regulatory protein alpha ligation induces macrophage nitric oxide production through JAK/STAT- and phosphatidylinositol 3-kinase/Rac1/NAPDH oxidase/H2O2-dependent pathways.

Signal regulatory protein alpha (SIRPalpha) is a glycoprotein receptor that recruits and signals via the tyrosine phosphatases SHP-1 and SHP-2. In macrophages SIRPalpha can negatively regulate the phagocytosis of host cells and the production of tumor necrosis factor alpha. Here we provide evidence that SIRPalpha can also stimulate macrophage activities, in particular the production of nitric oxide (NO) and reactive oxygen species. Ligation of SIRPalpha by antibodies or soluble CD47 triggers inducible nitric oxide synthase expression and production of NO. This was not caused by blocking negative-regulatory SIRPalpha-CD47 interactions. SIRPalpha-induced NO production was prevented by inhibition of the tyrosine kinase JAK2. JAK2 was found to associate with SIRPalpha in macrophages, particularly after SIRPalpha ligation, and SIRPalpha stimulation resulted in JAK2 and STAT1 tyrosine phosphorylation. Furthermore, SIRPalpha-induced NO production required the generation of hydrogen peroxide (H(2)O(2)) by a NADPH oxidase (NOX) and the phosphatidylinositol 3-kinase (PI3-K)-dependent activation of Rac1, an intrinsic NOX component. Finally, SIRPalpha ligation promoted SHP-1 and SHP-2 recruitment, which was both JAK2 and PI3-K dependent. These findings demonstrate that SIRPalpha ligation induces macrophage NO production through the cooperative action of JAK/STAT and PI3-K/Rac1/NOX/H(2)O(2) signaling pathways. Therefore, we propose that SIRPalpha is able to function as an activating receptor.

Neutrophils Inhibit Synthesis of Mineralized Extracellular Matrix by Human Bone Marrow-Derived Stromal Cells In Vitro.

Although controlled local inflammation is essential for adequate bone regeneration, several studies have shown that hyper-inflammatory conditions after major trauma are associated with impaired fracture healing. These hyper-inflammatory conditions include the trauma-induced systemic inflammatory response to major injury, open fractures, and significant injury to the surrounding soft tissues. The current literature suggests that increased or prolonged influx of neutrophils into the fracture hematoma may mediate impairment of bone regeneration after hyper-inflammatory conditions. The underlying mechanism remains unclear. We hypothesize that high neutrophil numbers inhibit synthesis of mineralized extracellular matrix (ECM) by bone marrow stromal cells (BMSCs). We therefore studied the effect of increasing concentrations of neutrophils on ECM synthesis by human BMSCs in vitro. Moreover, we determined how high neutrophil concentrations affect BMSC cell counts, as well as BMSC osteogenic activity determined by alkaline phosphatase (ALP) expression and ALP activity. Co-culture of BMSCs with neutrophils induced a 52% decrease in BMSC cell count (p < 0.01), a 64% decrease in the percentage of ALP+ cells (p < 0.001), a 28% decrease in total ALP activity (p < 0.01), and a significant decrease in the amount of mineralized ECM [38% decrease after 4 weeks (p < 0.05)]. Co-cultures with peripheral blood mononuclear cells and neutrophils within transwells did not induce a significant decrease in ALP activity. In conclusion, our data shows that a decreased amount of mineralized ECM became synthesized by BMSCs, when they were co-cultured with high neutrophil concentrations. Moreover, high neutrophil concentrations induced a decrease in BMSC cell counts and decreased ALP activity. Clarifying the underlying mechanism may contribute to development of therapies that augment bone regeneration or prevent impaired fracture healing after hyper-inflammatory conditions.

Osteoinduction by Ex Vivo Nonviral Bone Morphogenetic Protein Gene Delivery Is Independent of Cell Type.

Ex vivo nonviral gene delivery of bone inductive factors has the potential to heal bone defects. Due to their inherent role in new bone formation, multipotent stromal cells (MSCs) have been studied as the primary target cell for gene delivery in a preclinical setting. The relative contribution of autocrine and paracrine mechanisms, and the need of osteogenic cells, remains unclear. This study investigates the contribution of MSCs as producer of transgenic bone morphogenetic proteins (BMPs) and to what extent the seeded MSCs participate in actual osteogenesis. Rat-derived MSCs or fibroblasts (FBs) were cotransfected with pBMP-2 and pBMP-6 or pBMP-7 via nucleofection. The bioactivity of BMP products was shown through in vitro osteogenic differentiation assays. To investigate their role in new bone formation, transfected cells were seeded on ceramic scaffolds and implanted subcutaneously in rats. Bone formation was assessed by histomorphometry after 8 weeks. As a proof of principle, we also investigated the suitability of bone marrow-derived mononuclear cells and the stromal vascular fraction isolated from adipose tissue for a one-stage gene delivery strategy. Bone formation was induced in all conditions containing cells overexpressing BMP heterodimers. Constructs seeded with FBs transfected with BMP-2/6 and MSCs transfected with BMP-2/6 showed comparable bone volumes, both significantly higher than controls. Single-stage gene delivery proved possible and resulted in some bone formation. We conclude that bone formation as a result of ex vivo BMP gene delivery can be achieved even without direct osteogenic potential of the transfected cell type, suggesting that transfected cells mainly function as a production facility for osteoinductive proteins. In addition, single-stage transfection and reimplantation of cells appeared feasible, thus facilitating future clinical translation of the method.

Liposomal drug delivery in an in vitro 3D bone marrow model for multiple myeloma.

PURPOSE: Liposomal drug delivery can improve the therapeutic index of treatments for multiple myeloma. However, an appropriate 3D model for the in vitro evaluation of liposomal drug delivery is lacking. In this study, we applied a previously developed 3D bone marrow (BM) myeloma model to examine liposomal drug therapy. MATERIAL AND METHODS: Liposomes of different sizes (~75-200 nm) were tested in a 3D BM myeloma model, based on multipotent mesenchymal stromal cells, endothelial progenitor cells, and myeloma cells cocultured in hydrogel. The behavior and efficacy of liposomal drug therapy was investigated, evaluating the feasibility of testing liposomal drug delivery in 3D in vitro. Intracellular uptake of untargeted and integrin α4ß1 (very late antigen-4) targeted liposomes was compared in myeloma and supporting cells, as well as the effectivity of free and liposome-encapsulated chemotherapy (bortezomib, doxorubicin). Either cocultured myeloma cell lines or primary CD138+ myeloma cells received the treatments. RESULTS: Liposomes (~75-110 nm) passively diffused throughout the heterogeneously porous (~80-850 nm) 3D hydrogel model after insertion. Cellular uptake of liposomes was observed and was increased by targeting very late antigen-4. Liposomal bortezomib and doxorubicin showed increased cytotoxic effects toward myeloma cells compared with the free drugs, using either a cell line or primary myeloma cells. Cytotoxicity toward supporting BM cells was reduced using liposomes. CONCLUSION: The 3D model allows the study of liposome-encapsulated molecules on multiple myeloma and supporting BM cells, looking at cellular targeting, and general efficacy of the given therapy. The advantages of liposomal drug delivery were demonstrated in a primary myeloma model, enabling the study of patient-to-patient responses to potential drugs and treatment regimes.

Phosphate Functional Groups Improve Oligo[(Polyethylene Glycol) Fumarate] Osteoconduction and BMP-2 Osteoinductive Efficacy.

Off-the-shelf availability in large quantities, drug delivery functionality, and modifiable chemistry and mechanical properties make synthetic polymers highly suitable candidates for bone grafting. However, most synthetic polymers lack the ability to support cell attachment, proliferation, migration, and differentiation, and ultimately tissue formation. Incorporating anionic peptides into the polymer that mimics acidic proteins, which contribute to biomineralization and cellular attachment, could enhance bone formation. Therefore, this study investigates the effect of a phosphate functional group on osteoconductivity and BMP-2-induced bone formation in an injectable and biodegradable oligo[(polyethylene glycol) fumarate] (OPF) hydrogel. Three types of OPF hydrogels were fabricated using 0%, 20%, or 40% Bis(2-(methacryloyloxy)ethyl) phosphate creating unmodified OPF-noBP and phosphate-modified OPF-BP20 and OPF-BP40, respectively. To account for the osteoinductive effect of various BMP-2 release profiles, two different release profiles (i.e., different ratios of burst and sustained release) were obtained by varying the BMP-2 loading method. To investigate the osteoconductive effect of phosphate modification, unloaded OPF composites were assessed for bone formation in a bone defect model after 3, 6, and 9 weeks. To determine the effect of the hydrogel phosphate modification on BMP-2-induced bone formation, BMP-2 loaded OPF composites with differential BMP-2 release were analyzed after 9 weeks of subcutaneous implantation in rats. The phosphate-modified OPF hydrogels (OPF-BP20 and OPF-BP40) generated significantly more bone in an orthotopic defect compared to the unmodified hydrogel (OPF-noBP). Furthermore, the phosphate functionalized surface-enhanced BMP-2-induced ectopic bone formation regardless of the BMP-2 release profile. In conclusion, this study clearly shows that phosphate functional groups improve the osteoconductive properties of OPF and enhanced BMP-2-induced bone formation. Therefore, functionalizing hydrogels with phosphate groups by crosslinking monomers into the hydrogel matrix could provide a valuable method for improving polymer characteristics and holds great promise for bone tissue engineering.

Growth plate expression profiling: Large and small breed dogs provide new insights in endochondral bone formation.

The difference in the adult height of mammals, and hence in endochondral bone formation, is not yet fully understood and may serve to identify targets for bone and cartilage regeneration. In line with this hypothesis, the intra-species disparity between the adult height of Great Danes and Miniature Poodles was investigated at a transcriptional level. Microarray analysis of the growth plate of five Great Danes and five Miniature Poodles revealed 2,981 unique genes that were differentially expressed, including many genes with an unknown role in skeletal development. A signaling pathway impact analysis indicated activation of the cell cycle, extracellular matrix receptor interaction and the tight junction pathway, and inhibition of pathways associated with inflammation and the complement cascade. In additional validation steps, the gene expression profile of the separate growth plate zones for both dog breeds were determined. Given that the BMP signaling is known for its crucial role in skeletal development and fracture healing, and BMP-2 is used in orthopaedic and spine procedures for bone augmentation, further investigations concentrated on the BMP pathway.The canonical BMP-2 and BMP-6 signaling pathway was activated in the Great Danes compared to Miniature Poodles. In conclusion, investigating the differential expression of genes involved in endochondral bone formation in small and large breed dogs, could be a game changing strategy to provide new insights in growth plate development and identify new targets for bone and cartilage regeneration. © 2017 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 36:138-148, 2018.

Cellular immunotherapy on primary multiple myeloma expanded in a 3D bone marrow niche model.

Bone marrow niches support multiple myeloma, providing signals and cell-cell interactions essential for disease progression. A 3D bone marrow niche model was developed, in which supportive multipotent mesenchymal stromal cells and their osteogenic derivatives were co-cultured with endothelial progenitor cells. These co-cultured cells formed networks within the 3D culture, facilitating the survival and proliferation of primary CD138+ myeloma cells for up to 28 days. During this culture, no genetic drift was observed within the genomic profile of the primary myeloma cells, indicating a stable outgrowth of the cultured CD138+ population. The 3D bone marrow niche model enabled testing of a novel class of engineered immune cells, so called TEGs (αßT cells engineered to express a defined γδTCR) on primary myeloma cells. TEGs were engineered and tested from both healthy donors and myeloma patients. The added TEGs were capable of migrating through the 3D culture, exerting a killing response towards the primary myeloma cells in 6 out of 8 donor samples after both 24 and 48 hours. Such a killing response was not observed when adding mock transduced T cells. No differences were observed comparing allogeneic and autologous therapy. The supporting stromal microenvironment was unaffected in all conditions after 48 hours. When adding TEG therapy, the 3D model surpassed 2D models in many aspects by enabling analyses of specific homing, and both on- and off-target effects, preparing the ground for the clinical testing of TEGs. The model allows studying novel immunotherapies, therapy resistance mechanisms and possible side-effects for this incurable disease.

Endosteal and Perivascular Subniches in a 3D Bone Marrow Model for Multiple Myeloma.

The bone marrow microenvironment is the preferred location of multiple myeloma, supporting tumor growth and development. It is composed of a collection of interacting subniches, including the endosteal and perivascular niche. Current in vitro models mimic either of these subniches. By developing a model combining both niches, this study aims to further enhance the ability to culture primary myeloma cells in vitro. Also, the dependency of myeloma cells on each niche was studied. A 3D bone marrow model containing two subniches was created using 3D bioprinting technology. We used a bioprintable pasty calcium phosphate cement (CPC) scaffold with seeded osteogenic multipotent mesenchymal stromal cells (O-MSCs) to model the endosteal niche, and Matrigel containing both endothelial progenitor cells (EPCs) and MSCs to model the perivascular niche. Within the model containing one or both of the niches, primary CD138+ myeloma cells were cultured and analyzed for both survival and proliferation. The 3D bone marrow model with combined subniches significantly increasing the proliferation of CD138+ myeloma cells compared to both environments separately. The developed model showed an essential role of the perivascular niche over the endosteal niche in supporting myeloma cells. The developed model can be used to study the expansion of primary myeloma cells and their interactions with varying bone marrow subniches.