The genetic background determines material-induced bone formation through the macrophage-osteoclast axis.

Osteoinductive materials are characterized by their ability to induce bone formation in ectopic sites. Thus, osteoinductive materials hold promising potential for repairing bone defects. However, the mechanism of material-induced bone formation remains unknown, which limits the design of highly potent osteoinductive materials. Here, we demonstrated a genetic background link among macrophage polarization, osteoclastogenesis and material-induced bone formation. The intramuscular implantation of an osteoinductive material in FVB/NCrl (FVB) mice resulted in more M2 macrophages at week 1, more osteoclasts at week 2 and increased bone formation after week 4 compared with the results obtained in C57BL/6JOlaHsd (C57) mice. Similarly, in vitro, with a greater potential to form M2 macrophages, monocytes derived from FVB mice formed more osteoclasts than those derived from C57 mice. A transcriptomic analysis identified Csf1, Cxcr4 and Tgfbr2 as the main genes controlling macrophage-osteoclast coupling, which were further confirmed by related inhibitors. With such coupling, macrophage polarization and osteoclast formation of monocytes in vitro successfully predicted in vivo bone formation in four other mouse strains. Considering material-induced bone formation as an example of acquired heterotopic bone formation, the current findings shed a light on precision medicine for both bone regeneration and the treatment of pathological heterotopic bone formation.

Specific Targeting of Multiple Myeloma by Dual Split-signaling Chimeric Antigen Receptor T cells Directed against CD38 and CD138.

PURPOSE: The success of B-cell maturation antigen (BCMA)-specific chimeric antigen receptor (CAR) T cells illustrates the potential of this novel therapy for multiple myeloma. Nonetheless, broadening CAR T-cell therapy beyond BCMA requires inventive strategies as there are only a few multiple myeloma- or plasma cell-specific target antigens. We investigated the feasibility of achieving multiple myeloma specificity by dual-split CD38/CD138 CAR targeting, whereby the stimulatory and costimulatory signals for T-cell activation are split into two separate stimulatory (sCAR) and costimulatory CARs (cCAR). EXPERIMENTAL DESIGN: Using various combinations of CD38 and CD138 sCARs and cCARs with different affinities, we generated several dual-split CAR T cells and analyzed them for multiple myeloma-specific effector functions in vitro. The best-functioning CAR T cells were tested in vivo in a murine xenograft model. RESULTS: We found optimal designs of both CD38sCAR/CD138cCAR and CD138sCAR/CD38cCAR combinations, that effectively lysed multiple myeloma cells but spared single CD38- or CD138-positive healthy hematopoietic cells. While the CD38sCAR/CD138cCAR T cells achieved multiple myeloma-specific activity solely due to the low affinity of the CD38sCARs, the multiple myeloma-specific cytotoxicity, cytokine release, and proliferation of CD138sCAR/CD38cCAR T cells were established through a true combinatorial stimulatory and costimulatory effect. The most optimal combination comprised a low-affinity CD138sCAR combined with a high-affinity CD38cCAR. These CD138sCAR/CD38cCAR T cells also showed dual-antigen specific anti-multiple myeloma effects in vivo. Importantly, they were also effective against multiple myeloma cells from daratumumab pretreated patients with decreased CD38 expression levels. CONCLUSIONS: We demonstrate the possibility to specifically target multiple myeloma cells, even after CD38 targeted therapy, with carefully-designed dual-split CARs directed against CD38 and CD138.

Hypoxia Drives Material-Induced Heterotopic Bone Formation by Enhancing Osteoclastogenesis via M2/Lipid-Loaded Macrophage Axis.

Heterotopic ossification (HO) is a double-edged sword. Pathological HO presents as an undesired clinical complication, whereas controlled heterotopic bone formation by synthetic osteoinductive materials shows promising therapeutic potentials for bone regeneration. However, the mechanism of material-induced heterotopic bone formation remains largely unknown. Early acquired HO being usually accompanied by severe tissue hypoxia prompts the hypothesis that hypoxia caused by the implantation coordinates serial cellular events and ultimately induces heterotopic bone formation in osteoinductive materials. The data presented herein shows a link between hypoxia, macrophage polarization to M2, osteoclastogenesis, and material-induced bone formation. Hypoxia inducible factor-1α (HIF-1α), a crucial mediator of cellular responses to hypoxia, is highly expressed in an osteoinductive calcium phosphate ceramic (CaP) during the early phase of implantation, while pharmacological inhibition of HIF-1α significantly inhibits M2 macrophage, subsequent osteoclast, and material-induced bone formation. Similarly, in vitro, hypoxia enhances M2 macrophage and osteoclast formation. Osteoclast-conditioned medium enhances osteogenic differentiation of mesenchymal stem cells, such enhancement disappears with the presence of HIF-1α inhibitor. Furthermore, metabolomics analysis reveals that hypoxia enhances osteoclastogenesis via the axis of M2/lipid-loaded macrophages. The current findings shed new light on the mechanism of HO and favor the design of more potent osteoinductive materials for bone regeneration.

An off-the-shelf decellularized and sterilized human bone-ACL-bone allograft for anterior cruciate ligament reconstruction.

Approximately 1% of active individuals participating in sports rupture their anterior cruciate ligaments (ACL) every year, which is currently reconstructed using tendon autografts. Upon reconstruction, clinical issues of concern are ACL graft rupture, persistent knee instability, limited return to sports, and early onset of osteoarthritis (OA). This happens because tendon autografts do not have the same compositional, structural, and mechanical properties as a native ACL. To overcome these problems, we propose to use decellularized bone-ACL-bone allografts in ACL reconstruction (ACLR) as a mechanically robust, biocompatible, and immunologically safe alternative to autografts. Here, a decellularization protocol combined with sterilization using supercritical carbon dioxide (scCO2) was used to thoroughly decellularize porcine and human ACLs attached to tibial and femoral bone blocks. The specimens were named ultrACLean and their compositional, structural, and mechanical properties were determined. Our results indicate that: 1) decellularization of ultrACLean allografts leads to the removal of nearly 97% of donor cells, 2) ultrACLean has mechanical properties which are not different to native ACL, 3) ultrACLean maintained similar collagen content and decreased GAG content compared to native ACL, and 4) ultrACLean is not cytotoxic to seeded tendon-derived cells in vitro. Results from an in vivo pilot experiment showed that ultrACLean is biocompatible and elicits a moderate immunological response. In summary, ultrACLean has proven to be a mechanically competent and biocompatible graft with the potential to be used in ACLR surgery.

Combining a CAR and a chimeric costimulatory receptor enhances T cell sensitivity to low antigen density and promotes persistence.

Despite the high remission rates achieved using T cells bearing a chimeric antigen receptor (CAR) against hematogical malignancies, there is still a considerable proportion of patients who eventually experience tumor relapse. Clinical studies have established that mechanisms of treatment failure include the down-regulation of target antigen expression and the limited persistence of effective CAR T cells. We hypothesized that dual targeting mediated by a CAR and a chimeric costimulatory receptor (CCR) could simultaneously enhance T cell cytotoxicity and improve durability. Concomitant high-affinity engagement of a CD38-binding CCR enhanced the cytotoxicity of BCMA-CAR and CD19-CAR T cells by increasing their functional binding avidity. In comparison to second-generation BCMA-CAR or CD19-CAR T cells, double-targeted CAR + CD38-CCR T cells exhibited increased sensitivity to recognize and lyse tumor variants of multiple myeloma and acute lymphoblastic leukemia with low antigen density in vitro. In addition, complimentary costimulation by 4-1BB and CD28 endodomains provided by the CAR and CCR combination conferred increased cytokine secretion and expansion and improved persistence in vivo. The cumulatively improved properties of CAR + CCR T cells enabled the in vivo eradication of antigen-low tumor clones, which were otherwise resistant to treatment with conventional CAR T cells. Therefore, multiplexing targeting and costimulation through the combination of a CAR and a CCR is a powerful strategy to improve the clinical outcomes of CAR T cells by enhancing cytotoxic efficacy and persistence, thus preventing relapses of tumor clones with low target antigen density.

CD38-specific Chimeric Antigen Receptor Expressing Natural Killer KHYG-1 Cells: A Proof of Concept for an "Off the Shelf" Therapy for Multiple Myeloma.

Chimeric antigen receptor (CAR) T cells are highly successful in the treatment of hematologic malignancies. We recently generated affinity-optimized CD38CAR T cells, which effectively eliminate multiple myeloma (MM) cells with little or no toxicities against nonmalignant hematopoietic cells. The lack of universal donors and long manufacturing times however limit the broad application of CAR T cell therapies. Natural killer (NK) cells generated from third party individuals may represent a viable source of "off the shelf" CAR-based products, as they are not associated with graft-versus-host disease unlike allogeneic T cells. We therefore explored the preclinical anti-MM efficacy and potential toxicity of the CD38CAR NK concept by expressing affinity-optimized CD38CARs in KHYG-1 cells, an immortal NK cell line with excellent expansion properties. KHYG-1 cells retrovirally transduced with the affinity-optimized CD38CARs expanded vigorously and mediated effective CD38-dependent cytotoxicity towards CD38high MM cell lines as well as primary MM cells ex vivo. Importantly, the intermediate affinity CD38CAR transduced KHYG-1 cells spared CD38neg or CD38int nonmalignant hematopoietic cells, indicating an optimal tumor nontumor discrimination. Irradiated, short living CD38CAR KHYG-1 cells also showed significant anti-MM effects in a xenograft model with a humanized bone marrow-like niche. Finally, CD38CAR KHYG-1 cells effectively eliminated primary MM cells derived from patients who are refractory to CD38 antibody daratumumab. Taken together, the results of this proof-of-principle study demonstrate the potential value of engineering affinity-optimized CD38CARs in NK cells to establish effective anti-MM effects, with an excellent safety profile, even in patients who failed to response to most advanced registered myeloma therapies, such as daratumumab.

Coupling between macrophage phenotype, angiogenesis and bone formation by calcium phosphates.

The ability of calcium phosphate (CaP) materials to induce bone formation varies with their physicochemical properties, with surface topography as one of the most crucial triggers. In view of the natural wound healing processes (e.g., inflammation, angiogenesis, tissue formation and remodeling) initiated after surgical implantation, we here comparatively investigated the biological cascades occurring upon ectopic implantation of a tricalcium phosphate with submicron surface topography (TCP-S, osteoinductive) and a tricalcium phosphate with micron-scale topography (TCP-B, non-osteoinductive). In vitro, TCP-S facilitated M2 polarization of macrophages derived from a human leukemic cell line (THP-1) as shown by the enhanced secretion of TGF-ß and CCL18. Interestingly, the conditioned media of polarized M2 macrophages on TCP-S enhanced tube formation by human umbilical vein endothelial cells (HUVECs), while had no influence on the osteogenic differentiation of human bone marrow stromal cells (HBMSCs). Following an intramuscular implantation in canines, TCP-S locally increased typical M2 macrophage markers (e.g., IL-10) at week 1 to 3 and enhanced blood vessel formation after week 3 as compared to TCP-B. Bone formation was observed histologically in TCP-S 6 weeks after implantation, and bone formation was inhibited when an angiogenesis inhibitor (KRN633) was loaded onto TCP-S. No bone formation was observed for TCP-B. The data presented herein suggest strong links between macrophage polarization, angiogenesis and CaP-induced bone formation. STATEMENT OF SIGNIFICANCE: The ability of calcium phosphate (CaP) materials to induce bone formation varies with their physicochemical properties, and the key physicochemical properties relevant to CaP-induced bone formation have been outlined in the last two decades. However, the biological mechanism underlying this material-driven osteoinduction remains largely unknown. This manuscript presented demonstrates strong links between surface topography, macrophage polarization, angiogenesis and bone formation in CaP materials implanted in non-osseous sites. The finding may provide new clues for further exploring the possible mechanism underlying osteoinduction by CaP materials.

Macrophage polarization plays roles in bone formation instructed by calcium phosphate ceramics.

To investigate the roles of macrophages in material-instructed bone formation, two calcium phosphate (TCP) ceramics with the same chemistry but various scales of surface topography were employed in this study. After being implanted subcutaneously in FVB mice for 8 weeks, TCPs (TCP ceramics with submicron surface topography) gave rise to bone formation, while TCPb (TCP ceramics with micron surface topography) did not, showing the crucial role of surface topography scale in material-instructed bone formation. Depletion of macrophages with liposomal clodronate (LipClod) blocked such bone formation instructed by TCPs, confirming the role of macrophages in material-instructed bone formation. Macrophage cells (i.e. RAW 264.7 cells) cultured on TCPs in vitro polarized to tissue repair macrophages as evidenced by gene expression and cytokine production, while polarizing to pro-inflammatory macrophages on TCPb. Submicron surface topography of TCP ceramics directed macrophage polarization via PI3K/AKT pathways with the synergistic regulation of integrin ß1. Finally, the tissue repair macrophage polarization on TCPs resulted in osteogenic differentiation of mesenchymal stem cells in vitro. At early implantation in FVB mice, TCPs recruited more macrophages which polarized towards tissue repair macrophages with time. The present data demonstrate the important roles of macrophage polarization in bone formation instructed by calcium phosphate ceramics.

Bone Morphogenetic Protein 4 Gene Therapy in Mice Inhibits Myeloma Tumor Growth, But Has a Negative Impact on Bone.

Multiple myeloma is characterized by accumulation of malignant plasma cells in the bone marrow. Most patients suffer from an osteolytic bone disease, caused by increased bone degradation and reduced bone formation. Bone morphogenetic protein 4 (BMP4) is important for both pre- and postnatal bone formation and induces growth arrest and apoptosis of myeloma cells. BMP4-treatment of myeloma patients could have the potential to reduce tumor growth and restore bone formation. We therefore explored BMP4 gene therapy in a human-mouse model of multiple myeloma where humanized bone scaffolds were implanted subcutaneously in RAG2-/- γC-/-mice. Mice were treated with adeno-associated virus serotype 8 BMP4 vectors (AAV8-BMP4) to express BMP4 in the liver. When mature BMP4 was detectable in the circulation, myeloma cells were injected into the scaffolds and tumor growth was examined by weekly imaging. Strikingly, the tumor burden was reduced in AAV8-BMP4 mice compared with the AAV8-CTRL mice, suggesting that increased circulating BMP4 reduced tumor growth. BMP4-treatment also prevented bone loss in the scaffolds, most likely due to reduced tumor load. To delineate the effects of BMP4 overexpression on bone per se, without direct influence from cancer cells, we examined the unaffected, non-myeloma femurs by µCT. Surprisingly, the AAV8-BMP4 mice had significantly reduced trabecular bone volume, trabecular numbers, as well as significantly increased trabecular separation compared with the AAV8-CTRL mice. There was no difference in cortical bone parameters between the two groups. Taken together, BMP4 gene therapy inhibited myeloma tumor growth, but also reduced the amount of trabecular bone in mice. Our data suggest that care should be taken when considering using BMP4 as a therapeutic agent. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

The role of calcium phosphate surface structure in osteogenesis and the mechanisms involved.

Calcium phosphate (CaP) ceramics have been widely used for bone regeneration because of their ability to induce osteogenesis. Surface properties, including chemical composition and surface structure, are known to play a crucial role in osteoconduction and osteoinduction. This review systematically analyzes the effects of surface properties, in particular the surface structure, of CaP scaffolds on cell behavior and new bone formation. We also summarize the possible signaling pathways involved in the osteogenic differentiation of bone-related cells when cultured on surfaces with various structures in vitro. The significant immune response initiated by surface structure involved in osteogenic differentiation of cells is also discussed in this review. Taken together, the new biological principle for advanced biomaterials is not only to directly stimulate osteogenic differentiation of bone-related cells but also to modulate the immune response in vivo. Although the reaction mechanism responsible for bone formation induced by CaP surface structure is not clear yet, the insights on surface structure-mediated osteogenic differentiation and osteoimmunomodulation could aid the optimization of CaP-based biomaterials for bone regeneration. STATEMENT OF SIGNIFICANCE: CaP ceramics have similar inorganic composition with natural bone, which have been widely used for bone tissue scaffolds. CaP themselves are not osteoinductive; however, osteoinductive properties could be introduced to CaP materials by surface engineering. This paper systematically summarizes the effects of surface properties, especially surface structure, of CaP scaffolds on bone formation. Additionally, increasing evidence has proved that the bone healing process is not only affected by the osteogenic differentiation of bone-related cells, but also relevant to the the cooperation of immune system. Thus, we further review the possible signaling pathways involved in the osteogenic differentiation and immune response of cells cultured on scaffold surface. These insights into surface structure-mediated osteogenic differentiation and osteoimmunomodulated-based strategy could aid the optimization of CaP-based biomaterials.

Orbital floor repair using patient specific osteoinductive implant made by stereolithography.

The orbital floor (OF) is an anatomical location in the craniomaxillofacial (CMF) region known to be highly variable in shape and size. When fractured, implants commonly consisting of titanium meshes are customized by plying and crude hand-shaping. Nevertheless, more precise customized synthetic grafts are needed to meticulously reconstruct the patients' OF anatomy with better fidelity. As alternative to titanium mesh implants dedicated to OF repair, we propose a flexible patient-specific implant (PSI) made by stereolithography (SLA), offering a high degree of control over its geometry and architecture. The PSI is made of biodegradable poly(trimethylene carbonate) (PTMC) loaded with 40 wt % of hydroxyapatite (called Osteo-PTMC). In this work, we developed a complete work-flow for the additive manufacturing of PSIs to be used to repair the fractured OF, which is clinically relevant for individualized medicine. This work-flow consists of (i) the surgical planning, (ii) the design of virtual PSIs and (iii) their fabrication by SLA, (iv) the monitoring and (v) the biological evaluation in a preclinical large-animal model. We have found that once implanted, titanium meshes resulted in fibrous tissue encapsulation, whereas Osteo-PMTC resulted in rapid neovascularization and bone morphogenesis, both ectopically and in the OF region, and without the need of additional biotherapeutics such as bone morphogenic proteins. Our study supports the hypothesis that the composite osteoinductive Osteo-PTMC brings advantages compared to standard titanium mesh, by stimulating bone neoformation in the OF defects. PSIs made of Osteo-PTMC represent a significant advancement for patients whereby the anatomical characteristics of the OF defect restrict the utilization of traditional hand-shaped titanium mesh.

The role of ENPP1/PC-1 in osteoinduction by calcium phosphate ceramics.

In the past decade, calcium phosphate (CaP) ceramics have emerged as alternatives to autologous bone grafts for the treatment of large, critical-sized bone defects. In order to be effective in the regeneration of such defects, ceramics must show osteoinductive behaviour, defined as the ability to induce de novo heterotopic bone formation. While a set of osteoinductive CaP ceramics has been developed, the exact processes underlying osteoinduction, and the role of the physical and chemical properties of the ceramics, remain largely unknown. Previous studies have focused on the role of the transcriptome to shed light on the mechanism of osteoinduction at the mRNA level. To complement these studies, a proteomic analysis was performed to study the behaviour of hMSCs on osteoinductive and non-osteoinductive CaPs. The results of this analysis suggest that plasma cell glycoprotein 1 (PC-1), encoded by the ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) gene, plays a key role in the process of osteoinduction by CaP ceramics. Validation experiments have confirmed that indeed, the mRNA expression of ENPP1 and the production of PC-1 are higher on osteoinductive than on non-osteoinductive CaP ceramics, a trend that was also observed for other osteogenic markers such as bone morphogenetic protein 2 (BMP2) and osteopontin (OPN), but not for alkaline phosphatase (ALP). Our results also showed that the expression of PC-1 is restricted to those cells which are in direct contact with the CaP ceramic surface, plausibly due to the localised depletion of calcium and inorganic phosphate ions from the supersaturated cell culture medium as CaP crystallises on the ceramic surface. Replicating the surface of the osteoinductive ceramic in polystyrene resulted in a significant decrease in ENPP1 expression, suggesting that surface structural properties alone are not sufficient to induce ENPP1 expression. Finally, knocking down ENPP1 expression in hMSCs resulted in increased BMP2 expression, both at the mRNA and protein level, suggesting that ENPP1 is a negative regulator of BMP-2 signalling. Taken together, this study shows, for the first time, that ENPP1/PC-1 plays an important role in CaP-induced osteogenic differentiation of hMSCs and thus possibly osteoinduction by CaP ceramics. Furthermore, we have identified a crucial role for the interfacial (chemical) events occurring on the CaP ceramic surface in the process of osteoinduction. This knowledge can contribute to the development of new bone graft substitutes, with improved osteoinductive potential.

Combined CD28 and 4-1BB Costimulation Potentiates Affinity-tuned Chimeric Antigen Receptor-engineered T Cells.

PURPOSE: Targeting nonspecific, tumor-associated antigens (TAA) with chimeric antigen receptors (CAR) requires specific attention to restrict possible detrimental on-target/off-tumor effects. A reduced affinity may direct CAR-engineered T (CAR-T) cells to tumor cells expressing high TAA levels while sparing low expressing normal tissues. However, decreasing the affinity of the CAR-target binding may compromise the overall antitumor effects. Here, we demonstrate the prime importance of the type of intracellular signaling on the function of low-affinity CAR-T cells. EXPERIMENTAL DESIGN: We used a series of single-chain variable fragments (scFv) with five different affinities targeting the same epitope of the multiple myeloma-associated CD38 antigen. The scFvs were incorporated in three different CAR costimulation designs and we evaluated the antitumor functionality and off-tumor toxicity of the generated CAR-T cells in vitro and in vivo. RESULTS: We show that the inferior cytotoxicity and cytokine secretion mediated by CD38 CARs of very low-affinity (K d < 1.9 × 10-6 mol/L) bearing a 4-1BB intracellular domain can be significantly improved when a CD28 costimulatory domain is used. Additional 4-1BB signaling mediated by the coexpression of 4-1BBL provided the CD28-based CD38 CAR-T cells with superior proliferative capacity, preservation of a central memory phenotype, and significantly improved in vivo antitumor function, while preserving their ability to discriminate target antigen density. CONCLUSIONS: A combinatorial costimulatory design allows the use of very low-affinity binding domains (K d < 1 µmol/L) for the construction of safe but also optimally effective CAR-T cells. Thus, very-low-affinity scFvs empowered by selected costimulatory elements can enhance the clinical potential of TAA-targeting CARs.

Efficacy of a synthetic calcium phosphate with submicron surface topography as autograft extender in lapine posterolateral spinal fusion.

Posterolateral spinal fusion (PLF) is a common procedure in orthopedic surgery that is performed to fuse adjacent vertebrae to reduce symptoms related to spinal conditions. In the current study, a novel synthetic calcium phosphate with submicron surface topography was evaluated as an autograft extender in a validated rabbit model of PLF. Fifty-nine skeletally mature New Zealand white rabbits were divided into three groups and underwent single-level intertransverse process PLF at L4-5 using (1) autologous bone graft (ABG) alone or in a 1:1 combination with (2) calcium phosphate granules (ABG/BCPgranules ), or (3) granules embedded in a fast-resorbing polymeric carrier (ABG/BCPputty ). After 6, 9, and 12 weeks, animals were sacrificed and spinal fusion was assessed by manual palpation, Radiographs, micro-CT, mechanical testing (12 weeks only), histology, and histomorphometry. Based on all endpoints, all groups showed a gradual progression in bone formation and maturation during time, leading to solid fusion masses between the transverse processes after 12 weeks. Fusion assessments by manual palpation, radiography and histology were consistent and demonstrated equivalent fusion rates between groups, with high bilateral fusion rates after 12 weeks. Mechanical tests after 12 weeks indicated substantially lower range of motion for all groups, compared to non-operated controls. By histology and histomorphometry, the gradual formation and maturation of bone in the fusion mass was confirmed for each graft type. With these results, we describe the equivalent performance between autograft and a novel calcium phosphate material as an autograft extender in a rabbit model of PLF using an extensive range of evaluation techniques. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2080-2090, 2019.

Ectopic bone formation by aggregated mesenchymal stem cells from bone marrow and adipose tissue: A comparative study.

Tissue engineered constructs (TECs) based on spheroids of bone marrow mesenchymal stromal cells (BM-MSCs) combined with calcium phosphate microparticles and enveloped in a platelet-rich plasma hydrogel showed that aggregation of MSCs improves their ectopic bone formation potential. The stromal vascular fraction (SVF) and adipose-derived MSCs (ASCs) have been recognized as an interesting MSC source for bone tissue engineering, but their ectopic bone formation is limited. We investigated whether aggregation of ASCs could similarly improve ectopic bone formation by ASCs and SVF cells. The formation of aggregates with BM-MSCs, ASCs and SVF cells was carried out and gene expression was analysed for osteogenic, chondrogenic and vasculogenic genes in vitro. Ectopic bone formation was evaluated after implantation of TECs in immunodeficient mice with six conditions: TECs with ASCs, TECs with BM-MSC, TECs with SVF cells (with and without rhBMP2), no cells and no cells with rhBMP2. BM-MSCs showed consistent compact spheroid formation, ASCs to a lesser extent and SVF showed poor spheroid formation. Aggregation of ASCs induced a significant upregulation of the expression of osteogenic markers like alkaline phosphatase and collagen type I, as compared with un-aggregated ASCs. In vivo, ASC and SVF cells both generated ectopic bone in the absence of added morphogenetic proteins. The highest incidence of bone formation was seen with BM-MSCs (7/9) followed by SVF + rhBMP2 (4/9) and no cells + rhBMP2 (2/9). Aggregation can improve ectopic bone tissue formation by adipose-derived cells, but is less efficient than rhBMP2. A combination of both factors should now be tested to investigate an additive effect.

Tailoring surface nanoroughness of electrospun scaffolds for skeletal tissue engineering.

Electrospun scaffolds provide a promising approach for tissue engineering as they mimic the physical properties of extracellular matrix. Previous studies have demonstrated that electrospun scaffolds with porous features on the surface of single fibers, enhanced cellular attachment and proliferation. Yet, little is known about the effect of such topographical cues on cellular differentiation. Here, we aimed at investigating the influence of surface roughness of electrospun scaffolds on skeletal differentiation of human mesenchymal stromal cells (hMSCs). Scanning electron microscopy (SEM) and atomic force microscopy (AFM) analysis showed that the surface nanoroughness of fibers was successfully regulated via humidity control of the electrospinning environment. Gene expression analysis revealed that a higher surface roughness (roughness average (Ra)=71.0±11.0nm) supported more induction of osteogenic genes such as osteopontin (OPN), bone morphogenetic protein 2 (BMP2), and runt-related transcription factor 2 (RUNX2), while a lower surface roughness (Ra=14.3±2.5nm) demonstrated higher expression of other osteogenic genes including bone sialoprotein (BSP), collagen type I (COL1A1) and osteocalcin (OCN). Interestingly, a lower surface roughness (Ra=14.3±2.5nm) better supported chondrogenic gene expression of hMSCs at day 7 compared to higher surface roughness (Ra=71.0±11.0nm). Taken together, modulating surface roughness of 3D scaffolds appears to be a significant factor in scaffold design for the control of skeletal differentiation of hMSCs. STATEMENT OF SIGNIFICANCE: Tissue engineering scaffolds having specific topographical cues offer exciting possibilities for stimulating cells differentiation and growth of new tissue. Although electrospun scaffolds have been extensively investigated in tissue engineering and regenerative medicine, little is known about the influence of introducing nanoroughness on their surface for cellular differentiation. The present study provides a method to engineer electrospun scaffolds with tailoring surface nanoroughness and investigates the effect of such topographical cues on the process of human mesenchymal stromal cells differentiation into osteoblasts and chondrocytes linages. This strategy may help the design of nanostructured scaffolds for skeletal tissue engineering.

Mining for osteogenic surface topographies: In silico design to in vivo osseo-integration.

Stem cells respond to the physicochemical parameters of the substrate on which they grow. Quantitative material activity relationships - the relationships between substrate parameters and the phenotypes they induce - have so far poorly predicted the success of bioactive implant surfaces. In this report, we screened a library of randomly selected designed surface topographies for those inducing osteogenic differentiation of bone marrow-derived mesenchymal stem cells. Cell shape features, surface design parameters, and osteogenic marker expression were strongly correlated in vitro. Furthermore, the surfaces with the highest osteogenic potential in vitro also demonstrated their osteogenic effect in vivo: these indeed strongly enhanced bone bonding in a rabbit femur model. Our work shows that by giving stem cells specific physicochemical parameters through designed surface topographies, differentiation of these cells can be dictated.