Various approaches have been implemented to enhance bone regeneration, including the utilization of autologous platelet-rich plasma and bone morphogenetic protein-2. The objective of this study was to evaluate the impact of Marburg Bone Bank-derived bone grafts in conjunction with platelet-rich plasma (PRP), recombinant human bone morphogenetic protein-2 (rhBMP-2), and zoledronic acid (ZA) on osteogenesis within rabbit bone defects. Methodology. Bone defects (5mm in diameter) were created in the femurs of 96 male rabbits. The animals were allocated into five groups: (1) bone graft + PRP (BG + PRP), (2) bone graft + 5μg rhBMP-2 (BG + rhBMP-2), (3) bone graft + 5μg ZA (BG + ZA), (4) bone graft + 10μg rhBMP-2 + 5μg ZA (BG + rhBMP-2 + ZA), and (5) bone graft (BG). Marburg Bone Bank-processed human femoral head allografts were utilized for bone grafting. The rabbits were euthanized at 14-, 30-, and 60-days post-surgery, and their femurs underwent histopathological and histomorphometric assessments. Results. Histomorphometric analysis revealed significantly enhanced de novo osteogenesis within the bone allografts in the BG + PRP and BG + rhBMP-2 groups compared to the BG, BG + ZA, and BG + rhBMP-2 + ZA groups at 14 and 30 days (p < 0.05). However, on day 60, the BG + rhBMP-2 group exhibited elevated osteoclastic activity (early resorption). The local co-administration of ZA with thermally treated grafts impeded both bone graft resorption and new bone formation within the bone defect across all time points. The addition of ZA to BG + rhBMP-2 resulted in diminished osteogenic activity compared to the BG + rhBMP-2 group (p < 0.000). Conclusion. The study findings indicated that the combination of PRP and rhBMP-2 with Marburg bone grafts facilitates early-stage osteogenesis in bone defect healing. Incorporating ZA into the thermally treated bone graft hinders both graft resorption and de novo bone formation

Bone regeneration is an area of acute medical need, but its clinical success is hampered by the need to ensure rapid vascularization of osteogenic grafts. Vascular Endothelial Growth Factor (VEGF) is the master regulator of vascular growth and during bone development angiogenesis and osteogenesis are physiologically coupled through so-called angiocrine factors produced by blood vessels. However, how to exploit this process for therapeutic bone regeneration remains a challenge (1). Here we will describe recent work aiming at understanding the cross-talk between vascular growth and osteogenesis under conditions relevant for therapeutic bone regeneration. To this end we take advantage of a unique platform to generate controlled signalling microenvironments, by the covalent decoration of fibrin matrices with tunable doses and combinations of engineered growth factors. The combination of human osteoprogenitors and hydroxyapatite in these engineered fibrin matrices provides a controlled model to investigate how specific molecular signals regulate vascular invasion and bone formation in vivo. In particular, we found that:. 1). Controlling the distribution of VEGF protein in the microenvironment is key to recapitulate its physiologic function to couple angiogenesis and osteogenesis (2);. 2). Such coupling is exquisitely dependent on VEGF dose and on a delicate equilibrium between opposing effects. A narrow range of VEGF doses specifically activates Notch1 signaling in invading blood vessels, inducing a pro-osteogenic functional state called Type H endothelium, that promotes differentiation of surrounding mesenchymal progenitors. However, lower doses are ineffective and higher ones paradoxically inhibit both vascular invasion and bone formation (Figure 1) (3);. 3). Semaphorin3a (Sema3a) acts as a novel pro-osteogenic angiocrine factor downstream of VEGF and it mediates VEGF dose-dependent effects on both vascular invasion and osteogenic progenitor stimulation. In conclusion, vascularization of osteogenic grafts is not simply necessary in order to enable progenitor survival. Rather, blood vessels can actively stimulate bone regeneration in engineered grafts through specific molecular signals that can be harnessed for therapeutic purposes. Acknowledgements: This work was supported in part by the European Union Horizon 2020 Program (Grant agreement 874790 – cmRNAbone). For any figures and tables, please contact the authors directly

Stem cell therapy is an effective means to address the repair of large segmental bone defects. However, the intense inflammatory response triggered by the implants severely impairs stem cell differentiation and tissue regeneration. High-dose transforming growth factor β1 (TGF-β1), the most locally expressed cytokine in implants, inhibits osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and promotes tissue fibrosis, severely compromising the efficacy of stem cell therapy. Small molecule inhibitors of TGF-β1 can be used to ameliorate the osteogenic disorders caused by high concentrations of TGF-β1, but systemic inhibition of TGF-β1 function will cause strong adverse effects. How to find safe and reliable molecular targets to antagonize TGF-β1 remains to be elucidated. Orphan nuclear receptor Nr4a1, an endogenous inhibitory molecule of TGF-β1, suppresses tissue fibrosis, but its role in BMSC osteogenesis is unclear. We found that TGF-β1 inhibited Nr4a1 expression through HDAC4. Overexpression of Nr4a1 in BMSCs reversed osteogenic differentiation inhibited by high levels of TGF- β1. Mechanistically, RNA sequencing showed that Nr4a1 activated the ECM-receptor interaction and Hippo signaling pathway, which in turn promoted BMSC osteogenesis. In bone defect repair and fracture healing models, transplantation of Nr4a1-overexpressing BMSCs into C57BL/6J mice or treatment with the Nr4a1 agonist Csn-B significantly ameliorated inflammation-induced bone regeneration disorders. In summary, our findings confirm the endogenous inhibitory effect of Nr4a1 on TGF- β1 and uncover the effectiveness of Nr4a1 agonists as a therapeutic tool to improve bone regeneration, which provides a new solution strategy for the treatment of clinical bone defects and inflammatory skeletal diseases

In 2021 the bone grafting market was worth €2.72 billion globally. As allograft bone has a limited supply and risk of disease transmission, the demand for synthetic grafting substitutes (BGS) continues to grow while allograft bone grafts steadily decrease. Synthetic BGS are low in mechanical strength and bioactivity, inspiring the development of novel grafting materials, a traditionally laborious and expensive process. Here a novel BGS derived from sustainably grown coral was evaluated. Coral-derived scaffolds are a natural calcium carbonate bio-ceramic, which induces osteogenesis in bone marrow mesenchymal stem cells (MSCs), the cells responsible for maintaining bone homeostasis and orchestrating fracture repair. By 3D printing MSCs in coral-laden bioinks we utilise high throughput (HT) fabrication and evaluation of osteogenesis, overcoming the limitations of traditional screening methods. MSC and coral-laden GelXA (CELLINK) bioinks were 3D printed in square bottom 96 well plates using a CELLINK BIO X printer with pneumatic adapter Samples were non-destructively monitored during the culture period, evaluating both the sample and the culture media for metabolism (PrestoBlue), cytotoxicity (lactose dehydrogenase (LDH)) and osteogenic differentiation (alkaline phosphatase (ALP)). Endpoint, destructive assays used included qRT-PCR and SEM imaging. The inclusion of coral in the printed bioink was biocompatable with the MSCs, as reflected by maintained metabolism and low LDH release. The inclusion of coral induced osteogenic differentiation in the MSCs as seen by ALP secretion and increased RUNX2, collagen I and osteocalcin transcription. Sustainably grown coral was successfully incorporated into bioinks, reproducibly 3D printed, non-destructively monitored throughout culture and induced osteogenic differentiation in MSCs. This HT fabrication and monitoring workflow offers a faster, less labour-intensive system for the translation of bone substitute materials to clinic. Acknowledgements: This work was co-funded by Enterprise Ireland and Zoan Biomed through Innovation Partnership IP20221024

Osteomyelitis is an inflammatory condition accompanied by the destruction of bone and caused by an infecting microorganism. Open contaminated fractures can lead to the development of osteomyelitis of the fractured bone in 3-25% of cases, depending on fracture type, degree of soft-tissue injury, degree of microbial contamination, and whether systemic and/or local antimicrobial therapies have been administered. Untreated, infection will ultimately lead to non-union, chronic osteomyelitis, or amputation. We report a case series of 10 patients that presented with post-operative infected non-union of the distal femur with or without prior surgery. The cases were performed at Padmashree Dr. D. Y. Patil Hospital, Nerul, Navi Mumbai, India. All the patients’ consents were taken for the study which was carried out in accordance with the Helsinki Declaration. The methodology involved patients undergoing a two-stage procedure in case of no prior implant or a three-stage procedure in case of a previous implant. Firstly, debridement and implant removal were done. The second was a definitive procedure in form of knee arthrodesis with ring fixator and finally followed by limb lengthening surgery. Arthrodesis was planned in view of infection, non-union, severe arthritic, subluxated knee, stiff knee, non-salvage knee joint, and financial constraints. After all the patients demonstrated wound healing in 3 months along with good radiographic osteogenesis at the knee arthrodesis site, limb lengthening surgeries by tibial osteotomy were done to overcome the limb length discrepancy. Distraction was started and followed up for 5 months. All 10 patients showed results with sound knee arthrodesis and good osteogenesis at the osteotomy site followed by achieving the limb length just 1-inch short from the normal side to achieve ground clearance while walking. Our case series is unique and distinctive as it shows that when patients with infected nonunion of distal femur come with the stiff and non-salvage knee with severe arthritic changes and financial constraints, we should consider knee arthrodesis with Ilizarov ring fixator followed by limb lengthening surgery

Introduction. The incidences of fragility fractures, often because of osteoporosis, are increasing. Research has moved towards bioresorbable scaffolds that provide temporary mechanical stability and promote osteogenesis. This research aims to fabricate a 3D printed composite Poly (l-lactic-co-glycolic acid)-strontium doped tricalcium phosphate (PLGA-SrTCP) scaffold and evaluate in an in vitro co culture study containing osteoporotic donor cells. Method. PLGA, PLGA TCP, and PLGA SrTCP scaffolds were produced using Fused Filament Fabrication (FFF). A four-group 35-day cell culture study was carried out using human bone marrow derived mesenchymal stem cells (hMSCs) from osteoporotic and control donors (monoculture) and hMSCs & human monocytes (hMCs) (Co culture). Outcome measures were biochemical assays, PCR, and cell imaging. Cells were cultured on scaffolds that had been pre-degraded for six weeks at 47°C prior to drying and gamma sterilisation. Result. 3D printed scaffolds were successfully produced by FFF. All groups in the study supported cell attachment onto the scaffolds, producing extracellular matrices as well as evidence of osteoclast cell structures. Osteoporotic cells increased CTSK activity and CAII activity and decreased ALP activity compared to controls. In control cultures, the addition of bTCP and bTCP/Sr to the PLGA reduced TRAP5b, CAII and ALP activity compared to PLGA alone. The addition of Sr did not show any differences between donors. Conclusion. This study details suitability of 3D printed polymer scaffolds for use in bone tissue applications. Both composite and pure polymer scaffolds promote osteogenesis in vitro. The introduction of ceramic filler and ion doping does not beneficially effect osteogenic potential and can reduce its ability compared to pure polymer. This study suggests the behaviour of control and osteoporotic cells are different and that osteoporotic cells are more prone to bone resorption. Therefore, it is important to design bone scaffolds that are specific to the patient as well as to the region of fracture

There is still no consensus on which concentration of mesenchymal stem cells (MSCs) to use for promoting fracture healing in a rat model of long bone fracture. To assess the optimal concentration of MSCs for promoting fracture healing in a rat model. Wistar rats were divided into four groups according to MSC concentrations: Normal saline (C), 2.5 × 106 (L), 5.0 × 106 (M), and 10.0 × 106 (H) groups. The MSCs were injected directly into the fracture site. The rats were sacrificed at 2 and 6 자 post-fracture. New bone formation [bone volume (BV) and percentage BV (PBV)] was evaluated using micro-computed tomography (CT). Histological analysis was performed to evaluate fracture healing score. The protein expression of factors related to MSC migration [stromal cell-derived factor 1 (SDF-1), transforming growth factor-beta 1 (TGF-β1)] and angiogenesis [vascular endothelial growth factor (VEGF)] was evaluated using western blot analysis. The expression of cytokines associated with osteogenesis [bone morphogenetic protein-2 (BMP-2), TGF-β1 and VEGF] was evaluated using real-time polymerase chain reaction. Micro-CT showed that BV and PBV was significantly increased in groups M and H compared to that in group C at 6 wk post-fracture (P = 0.040, P = 0.009; P = 0.004, P = 0.001, respectively). Significantly more cartilaginous tissue and immature bone were formed in groups M and H than in group C at 2 and 6 wk post-fracture (P = 0.018, P = 0.010; P = 0.032, P = 0.050, respectively). At 2 wk post fracture, SDF-1, TGF-β1 and VEGF expression were significantly higher in groups M and H than in group L (P = 0.031, P = 0.014; P < 0.001, P < 0.001; P = 0.025, P < 0.001, respectively). BMP-2 and VEGF expression were significantly higher in groups M and H than in group C at 6 wk postfracture (P = 0.037, P = 0.038; P = 0.021, P = 0.010). Compared to group L, TGF-β1 expression was significantly higher in groups H (P = 0.016). There were no significant differences in expression levels of chemokines related to MSC migration, angiogenesis and cytokines associated with osteogenesis between M and H groups at 2 and 6 wk post-fracture. The administration of at least 5.0 × 106 MSCs was optimal to promote fracture healing in a rat model of long bone fractures

Chronic glucocorticoid use causes osteogenesis loss, accelerating the progression of osteoporosis. Histone methylation is shown to epigenetically increase repressive transcription, altering lineage programming of mesenchymal stem cells (MSC). This study is undertaken to characterize the action of histone demethylase UTX to osteogenic lineage specification of bone-marrow MSC and bone integrity upon glucocorticoid treatment. Bone-marrow MSC were incubated in osteogenic medium containing supraphysiological dexamethasone. Osteogenic gene expression and mineralized nodule formation were probed using RT-PCR and von Kossa staining. The enrichment of trimethylated lysine 27 at histone 3 (H3K27me3) in Dkk1 promoter was quantified using chromatin immunoprecipitation-PCR. Bone mass and trabecular morphometry in methylprednisolone-treated skeletons were quantified using microCT analysis. Supraphysiological dexamethasone decreased osteogenic genes Runx2 and osteocalcin expression and mineralized matrix production along with reduced UTX expression in MSC. Forced UTX expression attenuated the glucocorticoid-mediated loss of osteogenic differentiation, whereas UTX knockdown provoked osteogenesis loss and cytoplasmic oil overproduction. UTX demethylated H3K27 and reduced the glucocorticoid-mediated the H3K27 enrichment in Dkk1 promoter, reversing beta-catenin signal, but downregulating Dkk1 production by MSC. In vivo, treatment with UTX inhibitor GSK-J4 significantly suppressed bone mineral density, trabecular volume, and thickness along with porous trabecular, fatty marrow and disturbed beta-catenin/Dkk1 histopathology comparable with glucocorticoid-induced osteoporosis condition. This study offers a productive insight into how UTX protects MSC from methylated histone-mediated osteogenesis repression in the development of glucocorticoid-induced osteoporosis

Glucocorticoid excess is shown to deteriorate bone tissue integrity, increasing the risk of osteoporosis. Marrow adipogenesis at cost of osteogenesis is a prominent feature of this osteoporosis condition. Epigenetic pathway histone deacetylase (HDAC)-mediated histone acetylation regulates osteogenic activity and bone mass. This study is aimed to figure out what role of acetylated histone reader bromodomain-containing protein 4 (BRD4) did play in glucocorticoid-induced osteoporosis. Bone-marrow mesenchymal stem cells were incubated in osteogenic medium with or without 1 μM dexamethasone. Mineralized matrix and adipocyte formation were probed using von Kossa and Nile Red O staining, respectively. Osteogenic and adipogenic marker expression were quantified using RT-PCR. The binding of acetylated histone to promoter of transcription factors were detected using chromatin immunoprecipitation-PCR. Bone mineral density and microstructure in osteoporotic bone were quantified with microCT system. Glucocorticoid repressed osteogenic transcription factor Runx2 expression and mineralized matrix formation along with a low level of acetylated lysine 9 at histone 3 (H3K9ac), whereas BRD4 signaling and adipocytic formation were increased in cell cultures. BRD4 knockdown reversed the H3K9ac enrichment in Runx2 promoter and osteogenesis, but downregulated adipogenic differentiation. Silencing BRD4 attenuated H3K9ac occupancy in forkhead box P1 (Foxp1) relevant to lipid metabolism upon glucocorticoid stress. Foxp1 interference downregulated adipogenic activities of glucocorticoid-treated cells. In vivo, treatment with BRD4 inhibitor JQ-1 compromised the glucocorticoid-induced bone mineral density loss, spare trabecular structure, and fatty marrow, as well as improved biomechanical properties of bone tissue. Taken together, BRD4-mediated Foxp1 pathways drive mesenchymal stem cells shifting toward adipocytic cells rather than osteogenic cells to aggravates excessive marrow adipogenesis in the process of glucocorticoid-induced osteoporosis. Pharmacological inhibition of BRD4 signaling protects bone tissue from bone loss and fatty marrow in glucocorticoid-treated mice. This study conveys a new molecular insight into epigenetic regulation of osteogenesis and adipogenesis in osteoporotic skeleton and highlight the remedial effect of BRD4 inhibitor on glucocorticoid-induced bone loss

Recently, technologies to culture one or more cell types in three dimensions have attracted a great deal of attention in tissue engineering. Particularly, the improved viability, self-renewal capacity, and differentiation potential have been reported for stem cell spheroids. However, it is crucial to modulate spheroid functions with instructive signals to use multi-cellular spheroids in tissue engineering. We have been developing ECM-mimicking fibrous materials decorated with cell-instructive cues, which were incorporated within 3D stem cell spheroids to fine-tune their functions as modular building blocks for bottom-up tissue-engineering applications. In particular, we created composite spheroids of human adipose-derived stem cells (hADSCs) incorporating nanofibers coated with instructive signal of either transforming growth factor-β3 or bone morphogenetic growth factor-2 for chondrogenesis or osteogenesis of stem cells, respectively. The bilayer structure of osteochondral tissue was subsequently mimicked by cultivating each type of spheroid inside 3D-printed construct. The in vitro chondrogenic or osteogenic differentiation of hADSCs within the biphasic construct under general media was locally regulated by each inductive component. More importantly, hADSCs from each spheroid proliferated and sprouted to form the integrated tissue with interface of bone and cartilage tissue. This approach may be applied to engineer complex tissue with hierarchically organized structure

3D Printed polyether-ether-ketone (PEEK) has gained widespread use in clinical practice due to its excellent biocompatibility, biomechanical compatibility, and personalization. However, pre-printed PEEK implants are not without their flaws, including bioinert, optimization distortion of 3D printing digital model and prosthetic mismatching. Recent advancements in mechanical processing technology have made it possible to print bone implants with PEEK fused deposition, allowing for the construction of mechanically adaptable implants. In this study, we aimed to synthesize silanized polycitrate (PCS) via thermal polymerization and in situ graft it to PEEK surface to construct an elastomer coating for 3D printed PEEK implants (PEEK-PCS). This incorporation of PCS allows the implant to exhibit adaptive space filling ability and stress dispersal. In vivo and in vitro results, PEEK-PCS exhibited exceptional osseointegration and osteogenesis properties along with macrophage M2 phenotypic polarization, inflammatory factors reducing, promotion of osteogenic differentiation in bone marrow mesenchymal stem cells (BMSCs). Additionally, PEEK-PCS displays good autofluorescence properties in vitro and in vivo, with stable fluorescence for 14 days, suggesting potential bioimaging applications. The study confirms that PEEK in situ grafting with thermo-polymerized PCS elastomers is a viable approach for creating multifunctional (bone defect adaptation, bioimaging, immune regulation, and osseointegration) implants for bone tissue engineering

Bone regeneration is pivotal for the healing of fractures. In case this process is disturbed a non-union can occur. This can be induced by environmental factors such as smoking, overloading etc. Co-morbidities such as diabetes, osteoporosis etc. may be more intrinsic factors besides other disturbances in the process. Those pathways negatively influence the bone regeneration process. Several intrinsic signal transduction pathways (WNT, BMP etc.) can be affected. Furthermore, on the transcriptional level, important mRNA expression can be obstructed by deregulated miRNA levels. For instance, several miRNAs have been shown to be upregulated during osteoporotic fractures. They are detrimental for osteogenesis as they block bone formation and accelerate bone resorption. Modulating those miRNAs may revert the physiological homeostasis. Indeed, physiological fracture healing has a typical miRNA signature. Besides using molecular pathways for possible treatment of non-union fractures, providing osteogenic cells is another solution. In 5 clinical cases with non-union fractures with defects larger than 10 cm, successful administration of a 3D printed PCL-TCP scaffold with autologous bone marrow aspirate concentrate and a modulator of the pathogenetic pathway has been achieved. All patients recovered well and showed a complete union of their fractures within one year after start of the regenerative treatment. Thus, non-union fractures are a diverse entity. Nevertheless, there seem to be common pathogenetic disturbances. Those can be counteracted at several levels from molecular to cell. Compositions of those may be the best option for future therapies. They can also be used in a more personalized fashion in case more specific measurements such as miRNA signature and stem cell activity are applied

Bone defects require implantable graft substitutes, especially porous and biodegradable biomaterial for tissue regeneration. The aim of this study was to fabricate and assess a 3D-printed biodegradable hydroxyapatite/calcium carbonate scaffold for bone regeneration. Materials and methods:. A 3D-printed biodegradable biomaterial containing calcium phosphate and aragonite (calcium carbonate) was fabricated using a Bioplotter. The physicochemical properties of the material were characterised. The materials were assessed in vitro for cytotoxicity and ostegenic potential and in vivo in rat intercondylar Φ3mm bone defect model for 3 months and Φ5mm of mini pig femoral bone defects for 6 months. The results showed that the materials contained hydroxyapatite and calcium carbonate, with the compression strength of 2.49± 0.2 MPa, pore size of 300.00 ± 41mm, and porosity of 40.±3%. The hydroxyapatite/aragonite was not cytotoxic and it promoted osteogenic differentiation of human umbilical cord matrix mesenchymal stem cells in vitro. After implantation, the bone defects were healed in the treatment group whereas the defect of controlled group with gelatin sponge implantation remained non-union. hydroxyapatite/aragonite fully integrated with host bone tissue and bridged the defects in 2 months, and significant biodegradation was followed by host new bone formation. After implantation into Φ5mm femoral defects in mini pigs hydroxyapatite/aragonite were completed degraded in 6 months and fully replaced by host bone formation, which matched the healing and degradation of porcine allogenic bone graft. In conclusion, hydroxyapatite/aragonite is a suitable new scaffold for bone regeneration. The calcium carbonate in the materials may have played an important role in osteogenesis and material biodegradation

Recent researches indicate that both M1 and M2 macrophages play vital roles in tissue repair and foreign body reaction processes. In this study, we investigated the dynamics of M1 macrophages in the induced membrane using a mouse femur critical-sized bone defect model. The Masquelet method (M) and control (C) groups were established using C57BL/6J male mice (n=24). A 3mm-bone defect was created in the right femoral diaphysis followed by a Kirschner wire fixation, and a cement spacer was inserted into the defect in group M. In group C, the bone defect was left uninserted. Tissues around the defect were harvested at 1, 2, 4, and 6 weeks after surgery (n=3 in each group at each time point). Following Hematoxylin and eosin (HE) staining, immunohistochemical staining (IHC) was used to evaluate the CD68 expression as a marker of M1 macrophage. Iron staining was performed additionally to distinguish them from hemosiderin-phagocytosed macrophages. In group M, HE staining revealed a hematoma-like structure, and CD68-positive cells were observed between the spacer and fibroblast layer at 1 week. The number of CD68-positive cells decreased at 2 weeks, while they were observed around the new bone at 4 and 6 weeks. In group C, fibroblast infiltration and fewer CD68-positive cells were observed in the bone defect without hematoma-like structure until 2 weeks, and no CD68-positive cells were observed at 4 and 6 weeks. Iron staining showed hemosiderin deposition in the surrounding area of the new bone in both groups at 4 and 6 weeks. The location of hemosiderin deposition was different from that of macrophage aggregation. This study suggests that M1 macrophage aggregation is involved in the formation of induced membranes and osteogenesis and may be facilitated by the presence of spacers

Osteoporosis is a common problem in postmenopausal women and the elderly. 11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) is a bi-directional enzyme that primarily activates glucocorticoids (GCs) in vivo, which is a considerable potential target as treatment for osteoporosis. Previous studies have demonstrated its effect on osteogenesis, and our study aimed to demonstrate its effect on osteoclast activation. In vivo, we used 11β-HSD1 knock-off (KO) and C57BL6/J mice to undergo the ovariectomy-induced osteoporosis (OVX). In vitro, In vivo, We used 11β-HSD1 knockoff (KO) and C57BL6/J mice to undergo the ovariectomy-induced osteoporosis (OVX). In vitro, bone marrow-derived macrophages (BMM) and bone marrow mesenchymal stem cell (BMSC) of KO and C57BL6/J mice were extracted to test their osteogenic and osteoclastic abilities. We then created osteoclastic 11β-HSD1 elimination mice (Ctsk::11β-HSD1fl/fl) and treated them with OVX. Micro-CT analysis, H&E, immunofluorescence staining, and qPCR were performed. Finally, we conducted the high-throughput sequencing to find out 11β-HSD1 and osteoclast activation related genes. We collected 6w samples after modeling. We found that KO mice were resistant to loss of bone trabeculae. The same effect was observed in osteoclastic 11β-HSD1 elimination mice. Meanwhile, BVT-2733, a classic inhibitor of 11β-HSD1, inhibited the osteoclast effect of cells without affecting osteogenic effect in vitro. High-throughput sequencing suggested that glucocorticoid receptor (GR) may play a key role in the activation of osteoclasts, which was verified by immunofluorescence staining and WB in vivo and in vitro. In the process of osteoporosis, 11β-HSD1 expression of osteoclasts is abnormally increased, which may be a new target for inhibiting osteoclast activation and treating osteoporosis

Periosteal mesenchymal stem cells (PMSC) are an emerging niche of stem cells to enhance bone healing by tissue engineering process. They have to be differentiated into osteoprogenitors in order to synthesize new bone matrix. In vitro differentiation with specific differentiation medium (DM) is not exactly representative of what occurs in vivo. The interaction between PMSC and growth factors (GF) present in biological matrix is somewhat less understood. The goal of this study is to explore the possibility of spontaneous PMSC differentiation in contact with different biological matrices without DM. 500.000 porcine PMSC were seeded on 6-well plates and cultured with proliferation medium (PM). When reaching 80% confluence, biological samples (n=3) of demineralized bone matrix (DBM), decellularized porcine bone allograft (AOp), human bone allograft (AOh), human periosteum (HP) and human fascia lata (HFL) were added. Negative and positive control wells included cells with only PM or DM, respectively. The differentiation progress was assessed by Alizarin Red staining at days 7, 14 and 21. Bone morphogenetic protein content (BMP 2, 4, 5, 6, 7, 8, 9 and 11) of each sample was also investigated by western blot. Alizarin red highlighted bone nodules neoformation on wells containing AOp, AOh and DBM, like positive controls. HP and HFL wells did not show any nodules. These results are correlated to a global higher BMP expression profile in AOp than in HP and HFL but not statistically significant (p=0.38 and p>.99, respectively). The highest expression in each tissue was that of BMP2 and BMP7, which play an important role in osteoinduction. PMSC are well known to participate to bone formation but, despite BMP presence in HP and HFL, they did not permit to achieve osteogenesis alone. The bone contact seems to be essential to induce in vitro differentiation into osteoprogenitors

Successful application of patient derived cells to engineer vascularized bone grafts is often hampered by low cell numbers and lengthy in vitro expansion. With sound induced morphogenesis (SIM), local cell density enhancement was shown to improve microvasculature formation at lower cell concentration than conventional methods [1]. SIM takes advantage of hydrodynamic forces that act on cells to arrange them within a hydrogel. Following, we are evaluating the potential of cell-hydrogel biografts with high local cell density to improve the therapeutic efficacy in clinical scenarios such as anastomosis or bone formation within non-union fractures. To assess anastomosis, human umbilical vein endothelial cells (HUVEC) and human mesenchymal stromal cells (MSC) were mixed at a 1:1 ratio in PEG-based or Dextran-based hydrogels at a final concentration of 2×10. 6. cells×mL. -1. For ectopic bone formation, MSC were resuspended in PEG-based hydrogels at 2×10. 6. or 5×10. 6. cells×mL. -1. , with or without BMP-2. Cells were assembled into distinct patterns at a frequency of 60 Hz. Four biografts of 4 × 9 mm. 2. were implanted at the back of nude mice (total of 7 animals) and harvested after 2 or 8 weeks. Explants were fixed and imaged as whole constructs or embedded in paraffin for histological analysis. Upon explantation, microscopic evaluation indicated that HUVEC were retained within the PEG-hydrogel after 2 weeks and formed a pre-vascular network. In the second study, ectopic bone formation was more pronounced in areas of higher local cell density based on visual inspection. Ongoing experiments are further characterizing bone formation by micro-CT and histological evaluation. Our results indicate that local cell density enhancement by sound requires a lower initial cell concentration than conventional, static seeding methods to achieve comparable microvasculature structures or local osteogenesis

An increasing elderly population means joint replacement surgery numbers are projected to increase, with associated complications such as periprosthetic joint infections (PJI) also rising. PJI are particularly challenging due to antimicrobial resistant biofilm development on implant surfaces and surrounding tissues, with treatment typically involving invasive surgeries and systemic antibiotic delivery. Consequently, functionalisation of implant surfaces to prevent biofilm formation is a major research focus. This study characterises clinically relevant antimicrobials including gentamicin, clindamycin, daptomycin, vancomycin and caspofungin within a silica-based, biodegradable sol-gel coating for prosthetic devices. Antimicrobial activity of the coatings against clinically relevant microorganisms was assessed via disc diffusion assays, broth microdilution culture methods and the MBEC assay used to determine anti-biofilm activity. Human and bovine cells were cultured in presence of antimicrobial sol-gel to determine cytotoxicity using Alamar blue and antibiotic release was measured by LC-MS. Biodegradability in physiological conditions was assayed by FT-IR, ICP-MS and measuring mass change. Effect of degradation products on osteogenesis were studied by culturing mesenchymal stem cells in the presence of media in which sol-gel samples had been immersed. Antimicrobial-loaded coatings showed strong activity against a wide range of clinically relevant bacterial and fungal pathogens with no loss of activity from antibiotic alone. The sol-gel coating demonstrated controlled release of antimicrobials and initial sol-gel coatings showed no loss of viability on MSCs with gentamicin containing coatings. Current work is underway investigating cytotoxicity of sol-gel compositions against MG-63 cells and primary osteoblasts. This research forms part of an extended study into a promising antimicrobial delivery strategy to prevent PJI. The implant coating has potential to advance PJI infection prevention, reducing future burden upon healthcare costs and patient wellbeing

Osteoprogenitors on the inner layer of periosteum are the major cellular contributors to appositional bone growth and bone repair by callus formation. Previous work showed that periosteal-derived cells have little or no osteogenic activity under standard in vitro osteogenic culture conditions. This study was conducted to determine what growth factor(s) can activate periosteal osteogenic capacity. This study was conducted with IACUC approval. Periosteum from five equine donors was digested in collagenase for 3-4 hours at 37C. Isolated periosteal cells were maintained in DMEM/10% FBS medium and exposed to PDGF, Prostaglandin E2, BMP-2 and TGF-b3 at a range of concentrations for 72 hours. Changes in osteogenic gene expression (Runx2, OSX and ALP) were measured by qPCR. Periosteal cells were pre-treated with TGF-b3 or maintained in control medium were transferred into basal or osteogenic medium. Osteogenic status was assessed by Alizarin Red staining for mineralized matrix, ALP enzymatic activity and induction of osteogenic genes. PDGF, PgE2 and BMP-2 had little impact on expression of osteogenic markers by periosteal cells. In contrast, TGF-b3 stimulated significant increases in Osterix (over 100-fold) ALP expression (over 70-fold). Pre-treating periosteal cells with TGF-b3 for 72 hours stimulated rapid cell aggregation and aggregate mineralization once cells were transferred to osteogenic medium, while cells not exposed to TGF-b3 exhibited minimal evidence of osteogenic activity. This study indicate that TGF-b signaling is vital for periosteal osteogenic activity. Transient ‘priming’ of periosteal cells through TGF-b exposure was sufficient to activate subsequent osteogenesis without requiring ongoing growth factor stimulation. TGF beta ligands are secreted by many cell types, including periosteal progenitors and osteocytes, providing opportunities for both autocrine and paracrine pathways to regulate periosteal bone formation under homeostatic and reparative conditions

Wear debris from implant interfaces is the major factor leading to periprosthetic osteolysis. Fibroblast-like synoviocytes (FLSs) populate the intimal lining of the synovium and are in direct contact with wear debris. This study aimed to elucidate the effect of Ti particles as wear debris on human FLSs and the mechanism by which they might participate in the bone remodeling process during periprosthetic osteolysis. FLSs were isolated from synovial tissue from patients, and the condition medium (CM) was collected after treating FLSs with sterilized Ti particles. The effect of CM was analyzed for the induction of osteoclastogenesis or any effect on osteogenesis and signaling pathways. The results demonstrated that Ti particles could induce activation of the NFκB signaling pathway and induction of COX-2 and inflammatory cytokines in FLSs. The amount of RANL in the conditioned medium collected from Ti particle-stimulated FLSs (Ti CM) showed the ability to stimulate osteoclast formation. The Ti CM also suppressed the osteogenic initial and terminal differentiation markers for osteoprogenitors, such as alkaline phosphate activity, matrix mineralization, collagen synthesis, and expression levels of Osterix, Runx2, collagen 1α, and bone sialoprotein. Inhibition of the WNT and BMP signaling pathways was observed in osteoprogenitors after the treatment with the Ti CM. In the presence of the Ti CM, exogenous stimulation by WNT and BMP signaling pathways failed to stimulate osteogenic activity in osteoprogenitors. Induced expression of sclerostin (SOST: an antagonist of WNT and BMP signaling) in Ti particletreated FLSs and secretion of SOST in the Ti CM were detected. Neutralization of SOST in the Ti CM partially restored the suppressed WNT and BMP signaling activity as well as the osteogenic activity in osteoprogenitors. Our results reveal that wear debris-stimulated FLSs might affect bone loss by not only stimulating osteoclastogenesis but also suppressing the bone-forming ability of osteoprogenitors. In the clinical setting, targeting FLSs for the secretion of antagonists like SOST might be a novel therapeutic approach for preventing bone loss during inflammatory osteolysis