The occupation of the surface immunoglobulin antigen-binding site by oligomannose-type glycans (sIg-Mann) is a tumor-specific post-translational modification of classic follicular lymphoma (FL). SIg-Mann switches binding from antigen to dendritic cell-specific intercellular adhesion molecule 3 grabbing non-integrin (DC-SIGN), known to be expressed on interfollicular macrophages and FL-associated follicular dendritic cells (FDCs). The interaction with DC-SIGN induces reorganization of sIg-Mann in wider and less dense clusters than anti-Ig, consistent with inefficient DC-SIGN-induced endocytosis and a low-level intracellular signaling. However, ligand-specific cell clusters form between sIg-Mann-expressing lymphoma and DC-SIGN-expressing cells, raising a need to understand the functional consequences of the interaction of DC-SIGN with sIg-Mann on primary FL cells. This engagement induces adhesion of FL cells to vascular cell adhesion molecule-1 (VCAM-1) via B-cell receptor proximal kinases and actin regulators in a fashion similar to anti-Ig, but without initiating apoptosis in vitro. Instead, antibody blockade of sIg-Mann contact with DC-SIGN expressed on FDC-derived YK6/SIGN cells inhibits adhesion and survival of primary FL cells in vitro. These data highlight that the specific interaction with DC-SIGN induces FL cell adhesion to VCAM-1, likely allowing FL cell retention in the lymph node, and survival of the FL cells. Adhesion and survival are inhibited by an anti-DC-SIGN blocking antibody, indicating a new early therapeutic approach against FL retention and survival in adaptive tumor tissue niches.

OBJECTIVES:To examine the effectiveness of non-mineralised and pre-mineralised elastin-like recombinamer (ELR) membranes in guided bone regeneration (GBR) in a pre-clinical rabbit model. MATERIALS AND METHODS:Three pre-mineralised ELR membranes (D1, D2 and D8) and a non-mineralised (D0) membrane, along with a collagen membrane (C+) and an empty defect were randomly allocated in rabbit calvarial defects of critical size. Four weeks post-implantation the animals were euthanized and bone fill was assessed using micro CT and histological evaluation. RESULTS:Using micro CT, all ELR membranes led to similar bone density, as unassisted healing and healing using a resorbable collagen membrane. A trend for superior bone fill with the D2 pre-mineralised ELR membrane was observed. Histologically, at this early time-point the ELR membranes successfully guided GBR in the defects, similarly to the control collagen membrane, and additionally led to the formation of a thin woven bone layer at the top of the cranial defect bridging between the margins of the defect. CONCLUSIONS:As early as 4 weeks post-implantation, the ELR membranes successfully guided bone regeneration in this rabbit critical size calvarial model and a trend for superior regeneration was observed with pre-mineralised for 2 days ELR membranes. The ELR membranes led to the formation of a bony bridge parallel to the membrane suggesting bioactivity beyond occlusion of the defect. CLINICAL RELEVANCE:These bioactive alloplastic membranes may succesfully serve as alternatives to allogenic membranes in guided bone regeneration.

Autograft or metal implants are routinely used in skeletal repair. However, they fail to provide long-term clinical resolution, necessitating a functional biomimetic tissue engineering alternative. The use of native human bone tissue for synthesizing a biomimetic material ink for three-dimensional (3D) bioprinting of skeletal tissue is an attractive strategy for tissue regeneration. Thus, human bone extracellular matrix (bone-ECM) offers an exciting potential for the development of an appropriate microenvironment for human bone marrow stromal cells (HBMSCs) to proliferate and differentiate along the osteogenic lineage. In this study, we engineered a novel material ink (LAB) by blending human bone-ECM (B) with nanoclay (L, Laponite®) and alginate (A) polymers using extrusion-based deposition. The inclusion of the nanofiller and polymeric material increased the rheology, printability, and drug retention properties and, critically, the preservation of HBMSCs viability upon printing. The composite of human bone-ECM-based 3D constructs containing vascular endothelial growth factor (VEGF) enhanced vascularization after implantation in an ex vivo chick chorioallantoic membrane (CAM) model. The inclusion of bone morphogenetic protein-2 (BMP-2) with the HBMSCs further enhanced vascularization and mineralization after only seven days. This study demonstrates the synergistic combination of nanoclay with biomimetic materials (alginate and bone-ECM) to support the formation of osteogenic tissue both in vitro and ex vivo and offers a promising novel 3D bioprinting approach to personalized skeletal tissue repair.

BACKGROUND:Most patients with chronic lymphocytic leukaemia progress after treatment or retreatment with targeted therapy or chemoimmunotherapy and have limited subsequent treatment options. Response levels to the single-agent venetoclax in the relapsed setting is unknown. We aimed to assess venetoclax activity in patients with or without previous B-cell receptor-associated kinase inhibitor (BCRi) treatment. METHODS:This multicentre, open-label, single-arm, phase 3b trial (VENICE-1) assessed activity and safety of venetoclax monotherapy in adults with relapsed or refractory chronic lymphocytic leukaemia, stratified by previous exposure to a BCRi. Eligible participants were aged 18 years or older with previously treated relapsed or refractory chronic lymphocytic leukaemia. Presence of del(17p) or TP53 aberrations and previous BCRi treatment were permitted. Patients received 5-week ramp-up to 400 mg of oral venetoclax once daily and were treated for up to 108 weeks, with 2 years follow-up after discontinuation, or optional extended access. The primary activity endpoint was complete remission rate (complete remission or complete remission with incomplete marrow recovery) in BCRi-naive patients. Analyses used the intent-to-treat (ie, all enrolled patients, which coincided with those who received at least one dose of venetoclax). This study was registered with ClinicalTrials.gov, NCT02756611, and is complete. FINDINGS:Between June 22, 2016, and March 11, 2022, we enrolled 258 patients with relapsed or refractory chronic lymphocytic leukaemia (180 [70%] were male; 252 [98%] were White; 191 were BCRi-naive and 67 were BCRi-pretreated). Median follow-up in the overall cohort was 49·5 months (IQR 47·2-54·1), 49·2 months (47·2-53·2) in the BCRi-naive group, and 49·7 months (47·4-54·3) in the BCRi-pretreated group. Of 191 BCRi-naive patients, 66 (35%; 95% CI 27·8-41·8) had complete remission or complete remission with incomplete marrow recovery. 18 (27%; 95% CI 16·8-39·1) of 67 patients in the BCRi-pretreated group had complete remission or complete remission with incomplete marrow recovery. Grade 3 or worse treatment-emergent adverse events were reported in 203 (79%) and serious adverse events were reported in 136 (53%) of 258 patients in the overall cohort. The most common treatment-emergent adverse event was neutropenia (96 [37%]) and the most common and serious adverse event was pneumonia (21 [8%]). There were 13 (5%) deaths reported due to adverse events; one of these deaths (autoimmune haemolytic anaemia) was possibly related to venetoclax. No new safety signals were identified. INTERPRETATION:These data demonstrate deep and durable responses with venetoclax monotherapy in patients with relapsed or refractory chronic lymphocytic leukaemia, including BCRi-pretreated patients, suggesting that venetoclax monotherapy is an effective strategy for treating BCRi-naive and BCRi-pretreated patients. FUNDING:AbbVie.

Both chronic lymphocytic leukemias with unmutated (U-CLL) and mutated IGHV genes (M-CLL) are characterized by a variable state of anergy, defined by (auto)antigen-induced downmodulation of levels and function of the tumor IgM on the CLL cell surface (sIgM). The degree of anergy informs CLL progression and response to BTK inhibitors (BTKi), whereby patients with high sIgM have a more rapid progression and shorter duration of response to BTKi. Anergy appears particularly obvious in U-CLL selecting IGHV1-69(51p1) allele, which is infrequently used by normal B cells and represents ~30% of U-CLL. In IGHV1-69+ve U-CLL, sIgM level and function consistently and rapidly recover following (auto)antigen-mediated endocytosis. However, sIgM expression also requires translocation of the newly synthesized IgM by CD79B from endoplasmic reticulum to cell membrane. How this mechanism operates has not yet been investigated in CLL. The phenotypic, functional, and transcriptional consequences of sIgM engagement was investigated in single IGHV1-69+ve U-CLL and normal B cells (identified by anti-51p1 G6 monoclonal antibody F(ab)'). IGHV-IGHD-IGHJ analysis of >4500 single IGHV1-69(51p1) CLL cells from 3 patients documented a dominant U-IGHV1-69 rearrangement with low-level intraclonal heterogeneity in 0.8-1.9% cells, further supporting ongoing engagement with (auto)antigen. Single-cell RNAseq studies were performed in 3 IGHV1-69+ve U-CLL and 3 donors' normal B cells exposed ±anti-IgM F(ab')2 for 2 hours. UMAP analysis produced 11 clusters forming 3 major groups. The most notable group was composed of a cell cluster that overlapped distribution irrespective of anti-IgM stimulation, therefore it was termed as “unresponsive”. This group identified ~15% of the CLL clone, but was almost undetectable in the U-NBCs (<1%). The other 2 groups were an “unstimulated” group composed of cells that were not present in the stimulated sample, and a “stimulated” group composed of cell clusters that were present in the anti-IgM stimulated sample only. In contrast to the “unresponsive” group, the unstimulated and stimulated groups were evident in both the CLL and normal B-cells. In normal B cells, the BCR-associated transcripts IGHM, CD79A, and CD79B were high in the unstimulated group and were significantly downmodulated following the 2-hour anti-IgM engagement in vitro, indicating that antigen engagement promotes transcriptional suppression of BCR-associated transcripts. However, IGHM, CD79A, and CD79B levels were already low in the CLL “unstimulated” group, to levels not different from the normal and CLL “stimulated” groups. The observation that both unstimulated and stimulated CLL cells had low IGHM, CD79A, and CD79B transcript levels similar to in vitro stimulated normal B cells was a novel observation and indicated that antigen engagement has already occurred in CLL cells in vivo. Gene-set enrichment analysis of the 3 groups revealed that the “unresponsive” group was CLL-specific and had a profile associated with anergy. In particular, the “unresponsive” group had an increased Unfolded Protein Response (UPR), which provides an anti-apoptotic mechanism in addition to the BCL2 transcript overexpression (compared to normal B cells) which was evident in all 3 CLL groups. Specific analysis of the BCR-associated transcripts revealed that IGHM was recovering, with levels intermediate between the stimulated and unstimulated CLL cells, while CD79B was still undetectable at the 2-hour time point. This group of anergic cells are known to be the CLL cells with the most downmodulated sIgM glycoprotein. These data provide additional fundamental evidence of chronic (auto)antigen drive in CLL and a novel mechanism of antigen-mediated transcriptional control of sIgM expression levels. They identify the transcriptional characteristics of the CLL-specific anergic group. This group of cells will be insensitive to (auto)antigen and BTKi, while requiring targeting of the upregulated UPR pathway for complete eradication of the entire CLL clone. High BCL2 rescues these anergic cells from apoptosis and gives time for the IgM and CD79B transcripts to recover. Targeting these anergic B cells therapeutically will be key to complete therapeutic success in these CLL.

Introduction: The oligomannose-type glycans occupying the surface immunoglobulin antigen-binding site (sIg-Mann) are a tumor-founding post-translational requirement of classic follicular lymphoma (FL). These are universally acquired and a clonal necessity during the entire natural history of FL from the early stages throughout transformation into EZB-DLBCL (Chiodin G, Blood 2021, Odabashian M, Blood 2020). The low-affinity interaction of sIg-Mann with its specific ligand DC-SIGN distinguishes from conventional high-affinity antigen:Ig protein interactions for promoting prolonged low-level growth and prosurvival signals via PI3K/AKT and not endocytosis (Linley A, Blood 2015). However, the location and function of DC-SIGN remains to be further elucidated. Here, we investigated the hypothesis that the specific DC-SIGN:Ig-Mann interaction functions to promote retention of FL cells in tissue niches maintaining selective survival advantage. Methods: Immunofluorescence imaging was used to determine DC-SIGN histological location in primary FL lymph nodes. In WSU-FSCCL cells, DC-SIGN effects on sIg redistribution were measured by dSTORM; adhesion to VCAM-1 in presence/absence of inhibitors was measured by flow cytometry. Results: Immunofluorescence of lymph nodes from FL patients revealed that DC-SIGN was expressed on interfollicular CD163+ macrophages and, remarkably, on CD23+ follicular dendritic cells (FDC), claiming an influence on FL cell retention and survival. dSTORM revealed that DC-SIGN induced less dense and more diffuse surface Ig clusters compared to anti-Ig, reflecting an immature sIg redistribution unable to promote endocytosis and death. However, DC-SIGN efficiently induced adhesion of sIg-Mann+ve lymphoma cells to VCAM-1, known to be expressed on both lymph node macrophages and FDC, and inhibited migration towards SDF-1 in vitro. Blocking of DC-SIGN carbohydrate-recognition domain completely abrogated DC-SIGN-induced adhesion. Although intracellular signals were significantly lower, DC-SIGN induced levels of adhesion similar to those induced by anti-Ig. Adhesion to VCAM-1 was observed at concentrations from 20 µg/ml down to 20 ng/ml, even when AKT and ERK phosphorylation was not detectable. Either proximal inhibition of the PI3K/AKT pathway, or distal inhibition of ARP2/3, formin, and Cdc42 for lamellipodium formation at the surface membrane, suppressed adhesion. Conclusions: These results reveal an important mechanism of the DC-SIGN:sIg-Mann interaction that fine-tunes signals for membrane adaptation towards tumor cell adhesion, without promotion of cell death. This possibly facilitates FL cell retention in the lymph node protected tumor niche where selective growth and survival signals are maintained. Interrupting this interaction will be a novel way towards tumour-specific targeted therapy in FL patients. The research was funded by: This research was funded by Blood Cancer UK (grant 18009), Cancer Research UK (ECRIN-M3 accelerator award C42023/A29370, program C2750/A23669, and BTERP project C36811/A29101). D.T. was funded by the Eyles Cancer Immunology PhD scholarship, G.C. was funded by the Eyles Cancer Immunology Fellowship and the Southampton Cancer Immunology Centre Pump-priming award 2021). Keywords: Indolent non-Hodgkin lymphoma, Microenvironment, Targeting the Tumor Microenvironment Conflicts of interests pertinent to the abstract. F. Forconi Employment or leadership position: University of Siena Consultant or advisory role: Beigene Honoraria: Abbvie, Janssen-cilag, Beigene, Astra-Zeneca Research funding: Cancer Research UK Educational grants: Beigene, Abbvie Other remuneration: BC Platform

There is a wealth of data indicating human bone marrow contains skeletal stem cells (SSC) with the capacity for osteogenic, chondrogenic and adipogenic differentiation. However, current methods to isolate SSCs are restricted by the lack of a defined marker, limiting understanding of SSC fate, immunophenotype, function and clinical application. The current study applied single-cell RNA-sequencing to profile human adult bone marrow populations from 11 donors and identified novel targets for SSC enrichment. Spherical nucleic acids were used to detect these mRNA targets in SSCs. This methodology was able to rapidly isolate potential SSCs found at a frequency of <1 in 1,000,000 in human bone marrow, with the capacity for tri-lineage differentiation in vitro and ectopic bone formation in vivo. The current studies detail the development of a platform to advance SSC enrichment from human bone marrow, offering an invaluable resource for further SSC characterisation, with significant therapeutic impact therein.

The cells of virtually all CLL cases, including those with unmutated (U) Ig gene heavy-chain variable regions ( IGHV) of poorer prognosis (U-CLL), exhibit variable degree of anergy. This is defined by (auto)antigen-mediated low surface immunoglobulin M (sIgM), but not sIgD, expression and signaling capacity, both reversible in vitro. In U-CLL there is also a tumor-related highly asymmetric use of IGHV1-69 ( 51p1 allele) (~30% of all U-CLL vs ~1% of the normal B-cell repertoire), claiming selection by (auto)antigen. While the effect of (auto)antigen on sIgM down-modulation by endocytosis is well known, less is known about the biosynthetic mechanisms regulating sIgM expression. We investigated the phenotypic, functional, and transcriptional consequences of sIgM engagement in bulk and single cells of IGHV1-69+ve U-CLL, compared to IGHV1-69-ve U-CLL and non-tumor IGHV1-69+ve B cells (identified by G6 monoclonal antibody F(ab)‘). The functional consequences were measured by intracellular calcium [iCa 2+] mobilization assay following anti-IgM F(ab‘)2 engagement. Phenotypic and functional analyses of bulk IGHV1-69+ve U-CLL populations (n=32) documented homogeneously lower sIgM, but not sIgD, levels and signaling capacity compared to IGHV1-69-ve U-CLL (n=49). Also, sIgM engagement mobilized iCa 2+ more slowly than IGHV1-69-ve U-CLL. However, following in vitro culture in ‘antigen-free’ medium for 48 hours, sIgM levels and signaling capacity/speed recovered more rapidly in IGHV1-69+ve than IGHV1-69-ve U-CLL, indicating a more profound and dynamic control of Ig expression in the IGHV1-69+ve CLL cells. Single cell t-SNE plots of IGHV1-69+ve U-CLL (3 patients) and normal B cells (2 donors) exposed ±anti-IgM F(ab')2 identified 2 distinct unstimulated or anti-IgM stimulated B cell clusters, in which IGHM transcripts could also be compared. We found that the basal (unstimulated) IGHM transcript levels were downmodulated in CLL cells compared to normal B cells. However, while IGHM transcript levels reduced dramatically following 2 hours anti-IgM engagement in the stimulated normal B cells, further downmodulation was not visible in the stimulated CLL cells, suggesting that (auto)antigen induced IGHM transcript downmodulation had likely already occurred in those CLL cells in vivo. Combined kinetics studies of the IGHM transcripts by qPCR and sIgM levels in flow cytometry revealed that IGHM transcripts recovered in the CLL cells within 48 hours of culture in vitro. The increase of IGHM transcripts anticipated sIgM protein recovery of expression on the CLL cells in vitro. Instead, in the normal B cells there was no transcript or sIgM protein increase. These data in IGHV1-69+ve cells provide additional fundamental evidence of chronic (auto)antigen drive in CLL and a novel mechanism of antigen-mediated transcriptional control of sIgM levels. The antiapoptotic machinery provides valuable time to the circulating leukemic cells to resynthesize sIgM glycoprotein following downmodulation by endocytosis. Therefore, there is a danger of interrupting sIgM signaling by therapeutic approaches with BCR inhibitors alone. A coordinated inhibition of BCR signaling and cell survival is required to prevent sIgM recovery in CLL. We thank Dr Roy Jefferis, University of Birmingham, UK, for kindly providing the G6 hybridoma. Stuart Lanham and Luis Del Rio Fernandez should be considered joint first authors.

The B-cell receptor (BCR) is essential to the behavior of the majority of normal and neoplastic mature B cells. The identification in 1999 of the two major CLL subsets expressing unmutated immunoglobulin (Ig) variable region genes (U-IGHV, U-CLL) of pre-germinal center origin and poor prognosis, and mutated IGHV (M-CLL) of post-germinal center origin and good prognosis, ignited intensive investigations on structure and function of the tumor BCR. These investigations have provided fundamental insight into CLL biology and eventually the mechanistic rationale for the development of successful therapies targeting BCR signaling. U-CLL and M-CLL are characterized by variable low surface IgM (sIgM) expression and signaling capacity. Variability of sIgM can in part be explained by chronic engagement with (auto)antigen at tissue sites. However, other environmental elements, genetic changes, and epigenetic signatures also contribute to the sIgM variability. The variable levels have consequences on the behavior of CLL, which is in a state of anergy with an indolent clinical course when sIgM expression is low, or pushed towards proliferation and a more aggressive clinical course when sIgM expression is high. Efficacy of therapies that target BTK may also be affected by the variable sIgM levels and signaling and, in part, explain the development of resistance.

Chronic lymphocytic leukemia (CLL) cells have variably low surface IgM (sIgM) levels/signaling capacity, influenced by chronic antigen engagement at tissue sites. Within these low levels, CLL with relatively high sIgM (CLLhigh) progresses more rapidly than CLL with low sIgM (CLLlow). During ibrutinib therapy, surviving CLL cells redistribute into the peripheral blood and can recover sIgM expression. Return of CLL cells to tissue may eventually recur, where cells with high sIgM could promote tumor growth. We analyzed time to new treatment (TTNT) following ibrutinib in 70 patients with CLL (median follow-up of 66 months) and correlated it with pretreatment sIgM levels and signaling characteristics. Pretreatment sIgM levels correlated with signaling capacity, as measured by intracellular Ca2+ mobilization (iCa2+), in vitro (r = 0.70; P < .0001). High sIgM levels/signaling strongly correlated with short TTNT (P < .05), and 36% of patients with CLLhigh vs 8% of patients with CLLlow progressed to require a new treatment. In vitro, capacity of ibrutinib to inhibit sIgM-mediated signaling inversely correlated with pretherapy sIgM levels (r = -0.68; P = .01) or iCa2+ (r = -0.71; P = .009). In patients, sIgM-mediated iCa2+ and ERK phosphorylation levels were reduced by ibrutinib therapy but not abolished. The residual signaling capacity downstream of BTK was associated with high expression of sIgM, whereas it was minimal when sIgM expression was low (P < .05). These results suggested that high sIgM levels facilitated CLL cell resistance to ibrutinib in patients. The CLL cells, surviving in the periphery with high sIgM expression, include a dangerous fraction that is able to migrate to tissue and receive proliferative stimuli, which may require targeting by combined approaches.

The selective acquisition of oligomannose-type glycans at the antigen-binding site of the surface immunoglobulin (sIg-Mann) is a tumor-specific post-translational modification of all follicular lymphomas (FL). Our immunogenetic and structural analyses have revealed that these oligomannoses at N-glycosylation sites are universally acquired and persist during the entire natural history of FL from the early stages throughout transformation into EZB lymphoma (Chiodin G, Blood 2021). If a FL cell loses these sites, which is rare, the progeny disappears unless a new glycosylation site is acquired (Odabashian M, Blood 2020). These data indicate that sIg-Mann is a fundamental functional requirement for the entire clonal population of all FLs. The low-affinity interaction of sIg-Mann with its specific ligand dendritic cell-specific intercellular adhesion molecule 3 grabbing non-integrin (DC-SIGN), expressed on interfollicular M2-macrophages and follicular dendritic cells (FDC) of FL (RadtkeA, BioRxiv, 2022), distinguishes from conventional high-affinity antigen:Ig protein interactions for promoting prolonged low-level growth and prosurvival signals via AKT and MYC and not endocytosis (Linley A, Blood 2015). However, the function of DC-SIGN:Ig-Mann remains to be further elucidated. In this study, we investigated the hypothesis that the specific engagement of DC-SIGN:Ig-Mann interaction functions to promote homing, while maintaining selective growth and survival signals. We found that DC-SIGN induced adhesion of sIg-Mann+ve lymphoma cells to VCAM-1 and inhibited migration towards SDF-1 in vitro. Blocking of DC-SIGN with an anti-DC-SIGN antibody specific to the carbohydrate-recognition domain completely abrogated DC-SIGN-induced adhesion, confirming that adhesion was mediated by the selective interaction with the oligomannose of sIg-Mann. Although intracellular signals were significantly lower than those by soluble anti-IgM F(ab)2 (here used to mimic the binding of cross-linking polyvalent antigen to Ig protein), the level of adhesion induced by DC-SIGN was similar to that induced by anti-IgM F(ab)2. Adhesion to VCAM-1 was observed at concentrations from 20 µg/ml down to 20 ng/ml, even when AKT and ERK phosphorylation was not detectable by immunoblotting. However, either proximal inhibition of the PI3K/AKT pathway or distal inhibition of ARP2/3, formin, Rac and Cdc42 for lamellipodium formation at the surface membrane, suppressed adhesion. This suggested that Ig-Mann-mediated adhesion required both low-level intracellular signals and membrane adaptation (Valle-Argos, Sci Rep 2021). Remarkably, unlike anti-IgM F(ab)2, adhesion was specific to VCAM-1 but not to other stromal elements including fibronectin (FN). Instead, using the sIg-Mann+ve (IGHV4-34+ve) WSU-FSCCL cells, DC-SIGN appeared to mimic the interaction of monovalent ligands, including Fab fragments to either the Ig constant (anti-IgM Fab) or Ig variable region (9G4 Fab, which recognizes an epitope in the framework region 1 of IGHV4-34). Like DC-SIGN, Fab fragments induced selective adhesion to VCAM-1 and low-level signals via AKT, but neither endocytosis nor adhesion to FN. These results identify an important functional role of the DC-SIGN:sIg-Mann in mediating a signal that is tuned at the right threshold to promote selective and, in the absence of Ig endocytosis, persistent adhesion to environmental VCAM-1, determining homing of the FL tumor cells in the lymph node. This tumor-specific antigen-independent low-affinity DC:SIGN-Ig-Mann interaction contrasts to conventional high-affinity antigen:Ig protein interaction, which is prevented in FL (Schneider D, Blood 2015) and would instead lead to endocytosis and death. Therapeutic interruption of the interaction of DC-SIGN+ve cells with sIg-Mann+ve FL cells would provide a new way to displace the tumor cells from their prosurvival microenvironment.

Hormones have an important role in the regulation of fetal growth and development, especially in response to nutrient availability in utero. Using micro-CT and an electromagnetic three-point bend test, this study examined the effect of pancreas removal at 0.8 fraction of gestation on the developing bone structure and mechanical strength in fetal sheep. When fetuses were studied at 10 and 25 days after surgery, pancreatectomy caused hypoinsulinaemia, hyperglycaemia and growth retardation which was associated with low plasma concentrations of leptin and a marker of osteoclast activity and collagen degradation. In pancreatectomized fetuses compared to control fetuses, limb lengths were shorter, and trabecular (Tb) bone in the metatarsi showed greater bone volume fraction, Tb thickness, degree of anisotropy and porosity, and lower fractional bone surface area and Tb spacing. Mechanical strength testing showed that pancreas deficiency was associated with increased stiffness and a greater maximal weight load at fracture in a subset of fetuses studied near term. Overall, pancreas deficiency in utero slowed the growth of the fetal skeleton and adapted the developing bone to generate a more compact and connected structure. Maintenance of bone strength in growth-retarded limbs is especially important in a precocial species in preparation for skeletal loading and locomotion at birth.

Dataset to support article Enrichment of Skeletal Stem Cells from Human Bone Marrow Using Spherical Nucleic Acids ACS Nano 2021. https://doi.org/10.1021/acsnano.0c10683

Human bone marrow (BM)-derived stromal cells contain a population of skeletal stem cells (SSCs), with the capacity to differentiate along the osteogenic, adipogenic, and chondrogenic lineages, enabling their application to clinical therapies. However, current methods to isolate and enrich SSCs from human tissues remain, at best, challenging in the absence of a specific SSC marker. Unfortunately, none of the current proposed markers alone can isolate a homogeneous cell population with the ability to form bone, cartilage, and adipose tissue in humans. Here, we have designed DNA-gold nanoparticles able to identify and sort SSCs displaying specific mRNA signatures. The current approach demonstrates the significant enrichment attained in the isolation of SSCs, with potential therein to enhance our understanding of bone cell biology and translational applications.

There is a wealth of data indicating human bone marrow derived stromal cells (HBMSCs) contain the skeletal stem cell (SSC) with the potential to differentiate along the stromal osteogenic, adipogenic and chondrogenic lineages. However, despite these advances, current methods to isolate skeletal stem cells (SSCs) from human tissues have proved challenging as no single specific marker has been identified limiting understanding of SSC fate, immunophenotype and the widespread clinical application of these cells. While a number of cell surface markers can enrich for SSCs, none of the proposed markers, alone, provide a platform to isolate single cells with the ability to form bone, cartilage, and adipose tissue in humans. The current study details the application of oligonucleotide-coated nanoparticles, spherical nucleic acids (SNAs), to rapidly isolate human cells using mRNAs signatures detected in SSCs in real time, to identify stem and progenitor skeletal populations using single cell RNA sequencing. Based on scRNA-seq of samples from 11 patients, this method was able to identify novel targets for SSC enrichment, which were assessed in a total of 80 patients. This methodology was able to isolate potential SSCs found at a frequency of <1 in 1,000,000 in human bone marrow, with a capacity for tri-lineage differentiation in vitro . The current approach provides new targets and a platform to advance SSC isolation, enrichment with significant therapeutic impact therein.

Acellular soft hydrogels are not ideal for hard tissue engineering given their poor mechanical stability, however, in combination with cellular components offer significant promise for tissue regeneration. Indeed, nanocomposite bioinks provide an attractive platform to deliver human bone marrow stromal cells (HBMSCs) in three dimensions producing cell-laden constructs that aim to facilitate bone repair and functionality. Here we present the in vitro, ex vivo and in vivo investigation of bioprinted HBMSCs encapsulated in a nanoclay-based bioink to produce viable and functional three-dimensional constructs. HBMSC-laden constructs remained viable over 21 d in vitro and immediately functional when conditioned with osteogenic media. 3D scaffolds seeded with human umbilical vein endothelial cells (HUVECs) and loaded with vascular endothelial growth factor (VEGF) implanted ex vivo into a chick chorioallantoic membrane (CAM) model showed integration and vascularisation after 7 d of incubation. In a pre-clinical in vivo application of a nanoclay-based bioink to regenerate skeletal tissue, we demonstrated bone morphogenetic protein-2 (BMP-2) absorbed scaffolds produced extensive mineralisation after 4 weeks (p < 0.0001) compared to the drug-free and alginate controls. In addition, HBMSC-laden 3D printed scaffolds were found to significantly (p < 0.0001) support bone tissue formation in vivo compared to acellular and cast scaffolds. These studies illustrate the potential of nanoclay-based bioink, to produce viable and functional constructs for clinically relevant skeletal tissue regeneration.

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

Adverse programming of adult non-communicable disease can be induced by poor maternal nutrition during pregnancy and the periconception period has been identified as a vulnerable period. In the current study, we used a mouse maternal low-protein diet fed either for the duration of pregnancy (LPD) or exclusively during the preimplantation period (Emb-LPD) with control nutrition provided thereafter and postnatally to investigate effects on fetal bone development and quality. This model has been shown previously to induce cardiometabolic and neurological disease phenotypes in offspring. Micro 3D computed tomography examination at fetal stages Embryonic day E14.5 and E17.4, reflecting early and late stages of bone formation, demonstrated LPD treatment caused increased bone formation of relative high mineral density quality in males, but not females, at E14.5, disproportionate to fetal growth, with bone quality maintained at E17.5. In contrast, Emb-LPD caused a late increase in male fetal bone growth, proportionate to fetal growth, at E17.5, affecting central and peripheral skeleton and of reduced mineral density quality relative to controls. These altered dynamics in bone growth coincide with increased placental efficiency indicating compensatory responses to dietary treatments. Overall, our data show fetal bone formation and mineral quality is dependent upon maternal nutritional protein content and is sex-specific. In particular, we find the duration and timing of poor maternal diet to be critical in the outcomes with periconceptional protein restriction leading to male offspring with increased bone growth but of poor mineral density, thereby susceptible to later disease risk.

Nanoclays have generated interest in biomaterial design for their ability to enhance the mechanics of polymeric materials and impart biological function. As well as their utility as physical cross-linkers, clays have been explored for sustained localization of biomolecules to promote in vivo tissue regeneration. To date, both biomolecule-clay and polymer-clay nanocomposite strategies have utilised the negatively charged clay particle surface. As such, biomolecule-clay and polymer-clay interactions are set in competition, potentially limiting the functional enhancements achieved. Here, we apply specific bisphosphonate interactions with the positively charged clay particle edge to develop self-assembling hydrogels and functionalized clay nanoparticles with preserved surface exchange capacity. Low concentrations of nanoclay are applied to cross-link hyaluronic acid polymers derivatised with a pendant bisphosphonate to generate hydrogels with enhanced mechanical properties and preserved protein binding able to sustain, for over six weeks in vivo, the localized activity of the clinically licensed growth factor BMP-2.