Aims. The involvement of long non-coding RNA (lncRNA) in bone marrow mesenchymal stem cell (MSC) osteogenic differentiation during osteoporosis (OP) development has attracted much attention. In this study, we aimed to disclose how LINC01089 functions in human mesenchymal stem cell (hMSC) osteogenic differentiation, and to study the mechanism by which LINC01089 regulates MSC osteogenesis. Methods. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western blotting were performed to analyze LINC01089, miR-1287-5p, and heat shock protein family A (HSP70) member 4 (HSPA4) expression. The osteogenic differentiation of MSCs was assessed through alkaline phosphatase (ALP) activity, alizarin red S (ARS) staining, and by measuring the levels of osteogenic gene marker expressions using commercial kits and RT-qPCR analysis. Cell proliferative capacity was evaluated via the Cell Counting Kit-8 (CCK-8) assay. The binding of miR-1287-5p with LINC01089 and HSPA4 was verified by performing dual-luciferase reporter and RNA immunoprecipitation (RIP) experiments. Results. LINC01089 expression was reinforced in serum samples of OP patients, but it gradually diminished while hMSCs underwent osteogenic differentiation. LINC01089 knockdown facilitated hMSC osteogenic differentiation. This was substantiated by: the increase in ALP activity; ALP, runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and osteopontin (OPN) messenger RNA (mRNA) levels; and level of ARS staining. Meanwhile, LINC01089 upregulation resulted in the opposite effects. LINC01089 targeted miR-1287-5p, and the LINC01089 knockdown-induced hMSC osteogenic differentiation was repressed by miR-1287-5p depletion. HSPA4 is a downstream function molecule of the LINC01089/miR-1287-5p pathway; miR-1287-5p negatively modulated HSPA4 levels and attenuated its functional effects. Conclusion. LINC01089 negatively regulated hMSC osteogenic differentiation, at least in part, via governing miR-1287-5p/HSPA4 signalling. These findings provide new insights into hMSC osteogenesis and bone metabolism. Cite this article: Bone Joint Res 2024;13(12):779–789

Background Osteopontin (OPN), also known as bone sialoprotein I or secreted phosphoprotein 1, is a major non-collagenous bone matrix protein. A broad distribution has been detected in embryonic bone, osteoid, and fracture callus [Nomura et al. 2000] pointing out its central role in bone development and healing. It remains unclear weather mechanical conditions influence OPN synthesis and thereby osteoprogenitor cell differentiation. We investigated OPN mRNA-levels of bone marrow derived mesenchymal stem cells (bm-MSC) cultured in a previously described compression bioreactor (CBR) [Matziolis et al. under review] under dynamic compression (DC). Materials Bm-MSCs of 5 different individuals (mean age 61y) were seeded in a fibrin-alginate mix-matrix placed between two slices of lyophyliced cancellous bone. One group of constructs (n=10) underwent DC with 7kPa at 0.05 Hz, resulting in a matrix compression of 1mm at an heigh of 5mm, for 24 hours in the CBR. Constructs cultured under similar conditions but without DC served as control group (n=10). mRNA was extracted out of each construct after ending the DC, following the Trizol®-protocol. After cDNA-synthesis, GEArray Q series (Human Osteogenesis Gene Arrays) were performed and normalized versus GAPDH. Results We found an increase of OPN-expression in all dynamically compressed matrices. In the DC-group we found a mean of 5-fold increase of OPN mRNA compared to the control group (median: 0.43 vs. 0.09, p< 0.001). Discussion and Conclusion The results of this study demonstrate that an in vitro DC of bm-MSCs for 24 hours leads to an increased expression of OPN. We conclude that DC is an important element of early fracture healing by increasing the expression of OPN and thereby modulating progenitor cell differentiation immediately after mechanical instability caused by a fracture

Aims. Long non-coding RNAs (lncRNAs) act as crucial regulators in osteoporosis (OP). Nonetheless, the effects and potential molecular mechanism of lncRNA PCBP1 Antisense RNA 1 (PCBP1-AS1) on OP remain largely unclear. The aim of this study was to explore the role of lncRNA PCBP1-AS1 in the pathogenesis of OP. Methods. Using quantitative real-time polymerase chain reaction (qRT-PCR), osteogenesis-related genes (alkaline phosphatase (ALP), osteocalcin (OCN), osteopontin (OPN), and Runt-related transcription factor 2 (RUNX2)), PCBP1-AS1, microRNA (miR)-126-5p, group I Pak family member p21-activated kinase 2 (PAK2), and their relative expression levels were determined. Western blotting was used to examine the expression of PAK2 protein. Cell Counting Kit-8 (CCK-8) assay was used to measure cell proliferation. To examine the osteogenic differentiation, Alizarin red along with ALP staining was used. RNA immunoprecipitation assay and bioinformatics analysis, as well as a dual-luciferase reporter, were used to study the association between PCBP1-AS1, PAK2, and miR-126-5p. Results. The expression of PCBP1-AS1 was pre-eminent in OP tissues and decreased throughout the development of human bone marrow-derived mesenchymal stem cells (hBMSCs) into osteoblasts. PCBP1-AS1 knockdown and overexpression respectively promoted and suppressed hBMSC proliferation and osteogenic differentiation capacity. Mechanistically, PCBP1-AS1 sponged miR-126-5p and consequently targeted PAK2. Inhibiting miR-126-5p significantly counteracted the beneficial effects of PCBP1-AS1 or PAK2 knockdown on hBMSCs’ ability to differentiate into osteoblasts. Conclusion. PCBP1-AS1 is responsible for the development of OP and promotes its progression by inducing PAK2 expression via competitively binding to miR-126-5p. PCBP1-AS1 may therefore be a new therapeutic target for OP patients. Cite this article: Bone Joint Res 2023;12(6):375–386

Aims. To investigate whether idiopathic osteonecrosis of the femoral head (ONFH) is related to impaired osteoblast activities. Methods. We cultured osteoblasts isolated from trabecular bone explants taken from the femoral head and the intertrochanteric region of patients with idiopathic ONFH, or from the intertrochanteric region of patients with osteoarthritis (OA), and compared their viability, mineralization capacity, and secretion of paracrine factors. Results. Osteoblasts from the intertrochanteric region of patients with ONFH showed lower alkaline phosphatase (ALP) activity and mineralization capacity than osteoblasts from the same skeletal site in age-matched patients with OA, as well as lower messenger RNA (mRNA) levels of genes encoding osteocalcin and bone sialoprotein and higher osteopontin expression. In addition, osteoblasts from patients with ONFH secreted lower osteoprotegerin (OPG) levels than those from patients with OA, resulting in a higher receptor activator of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) ligand (RANKL)-to-OPG ratio. In patients with ONFH, osteoblasts from the femoral head showed reduced viability and mineralized nodule formation compared with osteoblasts from the intertrochanteric region. Notably, the secretion of the pro-resorptive factors interleukin-6 and prostaglandin E. 2. as well as the RANKL-to-OPG ratio were markedly higher in osteoblast cultures from the femoral head than in those from the intertrochanteric region. Conclusion. Idiopathic ONFH is associated with a reduced mineralization capacity of osteoblasts and increased secretion of pro-resorptive factors. Cite this article: Bone Joint Res 2021;10(9):619–628

Aims. The aim of this study was to develop a single-layer hybrid organic-inorganic sol-gel coating that is capable of a controlled antibiotic release for cementless hydroxyapatite (HA)-coated titanium orthopaedic prostheses. Methods. Coatings containing gentamicin at a concentration of 1.25% weight/volume (wt/vol), similar to that found in commercially available antibiotic-loaded bone cement, were prepared and tested in the laboratory for: kinetics of antibiotic release; activity against planktonic and biofilm bacterial cultures; biocompatibility with cultured mammalian cells; and physical bonding to the material (n = 3 in all tests). The sol-gel coatings and controls were then tested in vivo in a small animal healing model (four materials tested; n = 6 per material), and applied to the surface of commercially pure HA-coated titanium rods. Results. The coating released gentamicin at > 10 × minimum inhibitory concentration (MIC) for sensitive staphylococcal strains within one hour thereby potentially giving effective prophylaxis for arthroplasty surgery, and showed > 99% elution of the antibiotic within the coating after 48 hours. There was total eradication of both planktonic bacteria and established bacterial biofilms of a panel of clinically relevant staphylococci. Mesenchymal stem cells adhered to the coated surfaces and differentiated towards osteoblasts, depositing calcium and expressing the bone marker protein, osteopontin. In the in vivo small animal bone healing model, the antibiotic sol-gel coated titanium (Ti)/HA rod led to osseointegration equivalent to that of the conventional HA-coated surface. Conclusion. In this study we report a new sol-gel technology that can release gentamicin from a bioceramic-coated cementless arthroplasty material. In vitro, local gentamicin levels are in excess of what can be achieved by antibiotic-loaded bone cement. In vivo, bone healing in an animal model is not impaired. This, thus, represents a biomaterial modification that may have the potential to protect at-risk patients from implant-related deep infection. Cite this article: Bone Joint J 2021;103-B(3):522–529

Cyclooxygenase-2 (COX-2) activity is necessary for fracture healing to proceed normally. In most cell types, COX-2 is inductively expressed and acts in a coordinated pathway to produce prostaglandins, which affect many physiological processes including inflammation. In the fracture callus, however, COX-2 expression and the molecular and cellular processes affected by COX-2 activity remain poorly understood. Using LC-MS/MS and xMAP, we measured fracture callus prostaglandin and inflammatory cytokine levels. We found that inflammatory cytokines rapidly peaked after fracture before declining to normal levels by day 4 after fracture. However, callus prostaglandin levels did not peak until 4 days after fracture before returning to normal levels by day 10. We used immunohistochemistry to detected COX-2 expression in callus cells and found that COX-2 was expressed in callus chondrocytes and osteoclasts during endochondral ossification, including those osteoclasts at the callus chondro-osseous junction. Targeted deletion of the COX-2 gene (Ptgs2) in osteoclasts or in chondrocytes was found to delay fracture healing. Using cell-based experiments, we found that COX-2 expression could be induced in osteoclasts by osteopontin treatment, suggesting an integrin-dependent induction of COX-2 expression in osteoclasts. This was confirmed in vivo using mice lacking osteopontin or integrin ß3. Immunohistochemistry also showed abundant osteopontin expression at the callus chondro-osseous junction. The results indicate that COX-2 expression in osteoclasts is controlled by integrin-dependent signalling, that COX-2 expression in osteoclasts and chondrocytes is necessary for fracture healing to proceed normally, and that COX-2 expression in chondro-osseous junction osteoclasts may be induced by osteopontin-dependent signalling by chondrocytes

Critical size bone defects deriving from large bone loss are an unmet clinical challenge1. To account for disadvantages with clinical treatments, researchers focus on designing biological substitutes, which mimic endogenous healing through osteogenic differentiation promotion. Some studies have however suggested that this notion fails to consider the full complexity of native bone with respect to the interplay between osteoclast and osteoblasts, thus leading to the regeneration of less functional tissue2. The objective of this research is to assess the ability of our laboratory's previously developed 6-Bromoindirubin-3’-Oxime (BIO) incorporated guanosine diphosphate crosslinked chitosan scaffold in promoting multilineage differentiation of myoblastic C2C12 cells and monocytes into osteoblasts and osteoclasts1, 3, 4. BIO addition has been previously demonstrated to promote osteogenic differentiation in cell cultures5, but implementation of a co-culture model here is expected to encourage crosstalk thus further supporting differentiation, as well as the secretion of regulatory molecules and cytokines2. Biocompatibility testing of both cell types is performed using AlamarBlue for metabolic activity, and nucleic acid staining for distribution. Osteoblastic differentiation is assessed through quantification of ALP and osteopontin secretion, as well as osteocalcin and mineralization staining. Differentiation into osteoclasts is verified using SEM and TEM, qPCR, and TRAP staining. Cellular viability of C2C12 cells and monocytes was maintained when cultured separately in scaffolds with and without BIO for 21 days. Both scaffold variations showed a characteristic increase in ALP secretion from day 1 to 7, indicating early differentiation but BIO-incorporated sponges yielded higher values compared to controls. SEM and TEM imaging confirmed initial aggregation and fusion of monocytes on the scaffold's surface, but BIO addition appeared to result in smoother cell surfaces indicating a change in morphology. Late-stage differentiation assessment and co-culture work in the scaffold are ongoing, but initial results show promise in the material's ability to support multilineage differentiation. Acknowledgements: The authors would like to acknowledge the financial support of the Collaborative Health Research Program (CHRP) through CIHR and NSERC, as well as Canada Research Chair – Tier 1 in Regenerative Medicine and Nanomedicine, and the FRQ-S

Introduction and Objective. Osteonecrosis of the femoral head (ONFH) is an evolving and disabling condition that often leads to subchondral collapse in late stages. It is the underlying diagnosis for approximately 3%–12% of total hip arthroplasties (THAs) and the most frequent aetiology for young patients undergoing THA. To date, the pathophysiological mechanisms underlying ONFH remain poorly understood. In this study, we investigated whether ONFH without an obvious etiological factor is related to impaired osteoblast activities, as compared to age-matched patients with primary OA. Materials and Methods. We cultured osteoblasts isolated from trabecular bone explants taken from the femoral head of patients with ONFH and from intertrochanteric region of patients with ONFH or with OA and compared their in vitro mineralisation capacity and secretion of paracrine factors. Results. Compared to patients with OA, osteoblasts obtained from the intertrochanteric region of patients with ONFH showed reduced mineralisation capacity, which further decreased in osteoblasts from the femoral head of the same patient. Lower mineralisation of osteoblasts from patients with ONFH correlated with lower mRNA levels of genes encoding osteocalcin and bone sialoprotein and higher osteopontin expression. Osteoblasts from the intertrochanteric region of patients with ONFH secreted lower osteoprtegerin levels than those from patients with OA, resulting in a higher receptor activator of NF-κB ligand (RANKL)-to-osteoprotegerin (OPG) ratio. Notably, the RANKL-to-OPG ratio, as well as the secretion of the proresorptive factors interleukin-6 and prostaglandin E. 2. , was higher in osteoblasts from the femoral head of patients with ONFH than in those from the intertrochanteric region. Conclusions. ONFH is associated with a reduced mineralisation capacity of osteoblasts and increased secretion of proresorptive factors

The skeleton is tuned for sensing and responding to mechanical forces: a global bone strain moves the extra-cellular fluid through the lacunocanalicular network of compact bone, so gene expression of osteocytes is mechanically modulated by extra cellular fluid flow shear stress. Several studies showed that shear stress modulates bone cells gene expression: in vitro mechanical stimulation impacts the levels of alkaline phosphatase, cAMP, intracellular calcium, NO, prostaglandin E2, c-fos, COX-2, osteopontin and osteocalcin. Aim of this study is to investigate the effect of shear stress on SAOS-2 human osteoblasts proliferation, bone matrix production and mineralization, using a biostable polyurethane as scaffold and a perfusion bioreactor. Polyurethane scaffolds with an average porediameter of 624 micron were utilized. Scaffolds were sterilized and placed in to standard well-plates (condition A) and into a bioreactor with forced perfusion at a rate of 3 ml/min (condition B). Human osteosarcoma cell lineSAOS-2 was obtained from the ATCC and cultured in McCoy’s 5A modified medium. A suspension of 7′105 osteoblasts was added onto the top of each scaffold. Medium was changed every 3 days and sampled for osteopontin and-osteocalcin ELISA kits. After 16 days culture DNA and calcium contents were measured, light microscopy and SEM analysis were performed. In condition B, in comparison to A, we observed 33% higher cells proliferation, 12.6-fold higher osteopontin secretion, 99.6-foldhigher osteocalcin secretion and 8-fold higher calcium deposition. Microscopy observations revealed that in condition A osteoblasts were few with thin discontinuous extracellular matrix; in contrast shear stress induced 3D modeling of cells and matrix organization, so several cells were in multilayer with highly developed matrix and no surfaces were cell free. Statically cultured osteoblasts showed normal proliferation, but a very low matrix synthesis. Into bioreactor, which provides physiological levels of shear stress, the osteoblasts proliferated and showed increased metabolic activity

Bone remodelling is mediated through the synchronism of bone resorption (catabolism) by osteoclasts and bone formation (anabolism) by osteoblasts. Imbalances in the bone remodelling cycle represent an underling cause of metabolic bone diseases such as osteoporosis, where bone resorption exceeds formation (1). Current therapeutic strategies to repair osteoporotic bone fractures focus solely in targeting anabolism or supressing catabolism (2). However, these therapeutics do not reverse the structural damage present at the defect site, ultimately leading to impaired fracture healing, making the repair of osteoporotic fractures particularly challenging in orthopaedics. Herein, we focus on investigating a combined versatile pro-anabolic and anti-catabolic effect of Magnesium (Mg. 2+. ) to modulate bone cell behaviour (3), to develop an engineered biomimetic bio-instructive biomaterial scaffold structurally designed to enhance bone formation while impeding pathological osteoclast resorption activities to facilitate better bone healing and promote repair. Pre-osteoblasts MC3T3-E1 (OBs) and osteoclasts progenitors RAW 264.7 (OCs) cell lines were cultured in growth media exposed to increasing concentrations of MgCl. 2. (0, 0.5, 1, 10, 25 and 50mM) and the optimal concentration to concurrently promote the differentiation of OBs and inhibit the differentiation or funtion of RANKL-induced OCs was assessed. We next used Fluorescence Lifetime Imaging Microscopy to investigate changes in the metabolic pathways during OBs and OCs differentiation when exposed to increasing MgCl. 2. concentrations. We developed a range of magnesium-incorporated collagen scaffolds to permit the spatiotemporal release of Mg. 2+. within the established therapeutic window, and to investigate the behaviour of bone cells in a 3D environment. In our results, we reported an increase in the expression of the bone formation markers osteocalcin and osteopontin for OBs exposed to 10mM MgCl. 2. , and a significant downregulation of the osteoclast-specific markers TRAP and cathepsin K in RANKL-induced OCs differentiation when exposed to 25mM MgCl. 2. Moreover, 25mM MgCl. 2. induced changes in the energy metabolism of OCs from a predominantly oxidative phosphorylation towards a more glycolytic pathway suggesting a regulatory effect of Mg. 2+. in the underlying mechanisms of osteoclasts formation and function. The developed porous collagen-magnesium scaffolds significantly reduced the expression of early osteoclastogenic markers RANK and NFkB, and an elevated expression of the osteogenic markers Runx2 and Col1A1 was reported after 7 days. Our research to date has provided evidences to demonstrate the potential of Mg. 2+. to concurrently enhance osteogenesis while inhibiting osteoclastogenesis in vitro, potentially introducing new targets for developing therapies to repair osteoporotic bone fractures

Introduction: Bone grafts are frequently used in orthopaedic operations to augment bone healing. Autologous bone graft is the gold standard for osteogenesis, but the amount available from the patient’s iliac crest is often insufficient to fill the defect and donor site morbidity is a significant complication. Alternatively, allograft can be implanted into patients, however, processing is necessary to reduce the immunicity of the graft and the risk of transmission of infection, but this destroys osteoprogenitor cells and hence reduces the osteogenic properties of the graft. Mesenchymal stem cells (MSCs) are present in bone marrow and have the ability to differentiate into osteoblasts. Therefore our study examined the use of MCSs, from bone marrow, to enhance the osteogenic properties of allograft. Hypothesis: MSCs cultured on freeze-dried ethylene oxide treated bone allograft differentiate into osteoblasts, thereby increasing the osteogenic nature of the graft material. Method: After informed consent, bone marrow aspirates were taken from 10 patients during elective orthopaedic operations. MSCs were characterized using Stro-1 antibody and grown on freeze-dried ethylene oxide treated bone allograft in vitro. The hypothesis was tested on three groups of graft, with eight samples in each group. Firstly, freeze-dried ethylene oxide treated bone graft was tested (group 2). For a negative control, allograft was heated to 70°C to denature the osteogenic proteins (group 1). The final group tested the effect of additional osteogenic supplements (100nM dexamethasone, 0.05mM ascorbic acid and 10mM (-glycerol phosphate) on MSCs on allograft (group 3). Osteoblastic differentiation of MSCs was observed under scanning (SEM) and transmission (TEM) electron microscopy, and by measuring protein levels: alkaline phosphatase (ALP), osteopontin and type I pro-collagen over 14 days. Results: SEM confirmed that MSCs could be successfully cultured on bone allograft. Cells grown in groups 2 and 3 were characteristic of metabolically active osteoblasts and collagen extracellular matrix was observed under TEM. The amount of ALP protein produced by MSCs cultured in groups 2 and 3 increased significantly over 14 days (P< 0.05), but there was no increase in group 1. ALP, osteopontin and type I pro-collagen production was significantly greater for group 2 than for group 1 and for group 3 than for group 2 (P< 0.05). Discussion and Conclusions: ALP, type I pro-collagen and osteopontin proteins are known to be produced by osteoblasts during increasing cell maturation and the levels of each of these proteins increased significantly when MSCs were cultured on allograft for 14 days compared with the negative control. The addition of osteogenic supplements significantly increased production of these proteins. Furthermore, MSCs cultured in groups 2 and 3 produced extracellular collagen matrix. These results are consistent with allograft causing MSCs to differentiate into osteoblasts and that this differentiation increases with additional osteogenic supplements. This study confirms that MSCs, derived from autologous bone marrow, could be used to increase the osteogenic potential of allograft, thereby increasing bony healing in patients

Osteogenesis is key to fracture healing and osteointegration of implanted material. Modification of surfaces on a nanoscale has been shown to affect cell interaction with the material and can lead to preferential osteogenesis. We hypothesised that osteogenesis could be induced in a heterogeneous population of osteoprogenitor cells by circular nanopits on a material surface. Furthermore, we intended to assess any correlation between nanopit depth and osteoinductive potential. The desired topographies were embossed onto polycaprolactone (PCL) discs using pre-fabricated nickel shims. All pits had a diameter of 30μm and investigated pit depths were 80nm, 220nm and 333nm. Scanning electron microscopy confirmed successful embossing and planar controls were shown to be flat. A bone marrow aspirate was obtained from the femoral neck of a healthy adult undergoing a hip replacement. After establishing a culture, cells were seeded onto the PCL discs, suspended in basal media and incubated. Samples were fixed and stained after three and 28 days. Cells were stained for the adhesion molecule vinculin after three days. Lowest concentrations of vinculin were seen in the planar control group. Osteoprogenitor cells on the shallowest pits, 80nm, had larger and brighter adhesion complexes. After 28 days, osteocalcin and osteopontin expression were used as markers of cell differentiation into an osteoblastic phenotype. 220nm deep pits consistently produced cells with the highest concentrations of osteopontin (p = 0.017) with a similar trend of osteocalcin expression. Cells on all topographies had higher expression levels than the planar controls. We demonstrated stimulation of osteogenesis in a heterogeneous population of osteoprogenitor cells. This cell mix is similar to that present in fracture healing and after reaming for intramedullary devices or uncemented implants. All nanopit depths gave promising results with an optimum depth of 220nm after 28 days

Purpose of study and background. We have previously reported the development of injectable hydrogels for potential disc regeneration (NPgel) or bone formation which could be utilized in spinal fusion (Bgel). As there are multiple sources of mesenchymal stem cells (MSCs), this study investigated the incorporation of patient matched hMSCs derived from adipose tissue (AD) and bone marrow (BM) to determine their ability to differentiate within both hydrogel systems under different culture conditions. Methods and Results. Human fat pad and bone marrow derived MSCs were isolated from femoral heads of patients undergoing hip replacement surgery for osteoarthritis with informed consent. MSCs were encapsulated into either NPgel or Bgel and cultured for up to 6 weeks in 5% (NPgel) or 21% (Bgel) O. 2. Histology and immunohistochemistry was utilized to determine phenotype. Both fat and bone marrow derived MSCs, were able to differentiate into both cell lineages. NPgel culture conditions increased expression of matrix components such as collagen II and aggrecan and NP phenotypic markers FOXF1 and PAX1, whereas Bgel induced expression of collagen I and osteopontin, indicative of osteogenic differentiation. Conclusion. NPgel and Bgel were able to differentiate patient derived MSCs from different sources into both NP and osteogenic lineages, which may give rise to novel treatment strategies for IVD degeneration and spinal fusion, enabling choice for cell source according to patients' circumstances and needs. C Le Maitre and C Sammon hold a patent for the hydrogel described. Funded by MRC and Versus Arthritis

Aims: In a recent study, chemical microroughening of bioactive glass surface was shown to promote attachment of osteoblastic cells and osseointegration of porous bioactive glass implant. The current in vivostudy employed molecular biologic techniques to clarify the osteogenic effects of smooth and microrough glass surfaces. Methods:Using a rat model, a portion of the medullary canal in the proximal tibia was evacuated and filled with microroughened or smooth bioactive glass microspheres. The primary bone healing response and subsequent remodelling were analysed at 1, 2, and 8 weeks, respectively. The expression of various genes for the bone matrix components (type I collagen, osteocalcin, osteopontin, osteonectin) and proteolytic enzymes (cathepsin K, MMP-9) were determined by Northern analysis. Results: The microroughened bioactive glass microspheres were found to induce higher mRNA levels for osteopontin and lower levels for osteonectin at 2 weeks after operation when compared to smooth control micropheres. At 8 weeks, the MMP-9 expression levels were significantly higher with microroughened bioactive glass microspheres. Conclusion: Microroughening of the bioactive glass surface triggered temporal changes in the expression of specific genes

Summary Statement. A biomimetic tissue engineering strategy involving culture on bone scaffolds in perfusion bioreactors allows the construction of stable, viable, patient-specific bone-like substitutes from human induced pluripotent stem cells. Introduction. Tissue engineering of viable bone substitutes represents a promising therapeutic strategy to mitigate the burden of bone deficiencies. Human induced pluripotent stem cells (hiPSCs) have an excellent proliferation and differentiation capacity, and represent an unprecedented resource for engineering of autologous tissue grafts, as well as advanced tissue models for biological studies and drug discovery. A major challenge is to reproducibly expand, differentiate and organize hiPSCs into mature, stable tissue structures. Based on previous studies (1,2,3), we hypothesised that the culture conditions supporting bone tissue formation from adult human mesenchymal stem cells (hMSCs) and human embryonic stem cell (hESC)-derived mesenchymal progenitors could be translated to hiPSC-derived mesenchymal progenitors. Our objectives were to: 1. Derive and characterise mesenchymal progenitors from hiPSC lines. 2. Engineer bone substitutes from progenitor lines exhibiting osteogenic potential in an osteoconductive scaffold – perfusion bioreactor culture model. 3. Assess the molecular changes associated with the culture of hiPSC-progenitors in perfusion bioreactors, and evaluate the stability of engineered bone tissue substitutes in vivo. Methods. hESC and hiPSC lines (derived using retroviral vectors, Sendai virus and episomal vectors) were karyotyped, characterised for pluripotency and induced into the mesenchymal lineage. Mesenchymal progenitors were evaluated for growth potential, expression of surface markers and differentiation potential. Progenitors exhibiting osteogenic potential were cultured on decellularised bovine bone scaffolds in perfusion bioreactors for 5 weeks as previously (3). Global gene expression profiles were evaluated prior and after bioreactor culture. Bone development was investigated using biochemical and histological methods, and by micro-computed tomography (μCT) imaging over the duration of bioreactor culture and after 12-week subcutaneous implantation in immunodeficient mice. Results. Progenitors with high proliferation potential, expressing typical mesenchymal surface antigens were successfully derived from three hiPSC lines. Differences in mesenchymal surface antigens expression and global gene expression profiles of progenitors from different lines corresponded to their differentiation abilities toward the osteogenic, chondrogenic and adipogenic lineages. Bioreactor culture yielded constructs with significantly higher cellularity, AP activity and osteopontin release into the culture medium as compared to static culture. Dense bone matrix formation was evidenced by the positive staining of collagen, osteopontin, bone sialoprotein and osteocalcin. In comparison, static culture yielded constructs with uniformly distributed cells, however tissue formation was scarce. μCT revealed a significant increase in bone structural parameters, evidencing mineralization of the deposited bone tissue during the 5-week culture in bioreactors. Osteogenesis and bone tissue formation were comparable between hESCs, hiPSCs and hMSCs (3). Bioreactor cultivation resulted in repression of genes involved in proliferation and tumorigenesis, and upregulation of genes associated with osteogenesis and bone development. Engineered bone tissue displayed stable phenotype after 12-week implantation in vivo, with cells of human origin, ingrowing vasculature and osteoclasts, suggesting an initiation of tissue remodeling. Discussion/Conclusion. Our biomimetic strategy opens the possibility to construct an unlimited quantity of patient-specific bone grafts for personalised applications, and to generate qualified experimental models to study bone biology under normal and pathological conditions, as well as test new drugs using selected pools of hiPSC lines

Introduction: Human bone marrow stromal stem cells(BMSSC’s) have the ability to differentiate into a variety of mesenchymal cell types including osteoblasts, fibroblasts, adipiocytes and myocytes. These stromal cells are involved in the process of bone formation during the healing of fractures. Collagen lyophilisate is a sterile extract of bovine demineralised bone matrix. This material contains proteins removed from bone that may control the differentiation of osteoblasts from BMSSC’s. These proteins are localised within collagen type 1. The aim of this study was to determine the effects of collagen lyophilisate on the osteogenic differentiation of bone marrow stromal stem cells cultured in vitro. Methods: Bone marrow was aspirated from the iliac crest of a human donor who was undergoing an unrelated elective orthopadic surgical procedure. Stromal stem cells were isolated from marrow, cultured and then characterised using immunofluorescent antibodies to Stro −1, a stromal stem cell marker. 3x10. 4. BMSSC’s were seeded into each of 3 culture wells and incubated with standard growth medium or standard medium with collagen lyophilisate diluted 1:50 or 1:100. Cells were cultured for a maximum duration of 30 days. At selected time intervals until day 30, osteogenic differentiation was assessed by determination of alkaline phosphatase, osteopontin, pro collagen carboxyterminal (type 1 collagen synthesis) and calcium in cultures using specific assays. Results: Cells cultured in collagen lyophilisate displayed a polygonal morphology early in the culture period and later formed complex aggregates. Cells in control cultures maintained a fibroblstic morphology until confluence. On day 21 alkaline phosphatase activity was significantly higher in collagen lyophilisate containing cultures than control cultures. Osteopontin levels were not enhanced in the collagen lyophilisate containing cultures. Type 1 collagen synthesis was higher in the collagen lyophilisate 1:50 group than all other groups at day 14. No differences in type 1 collagen synthesis were detected between cultures at other time periods. Calcium was not detected in any of the control cultures for the duration of the culture period. In contrast, calcium was detected in collagen lyophilisate containing cultures on day 15. Conclusion: Collagen lyophilisate resulted in changes in cellular morphology and arrangement. The ability of collagen lyophilisate to enhance alkaline phosphatase activity, increase collagen type 1 expression and stimulate the deposition of calcium in stromal stem cell cultures provides evidence that it has osteogenic properties

Since the development of biomimetic and ceramic bone reconstructive in the early 1970, these specialised bioreactors intended for bone or cartilage regeneration have come a long way in trying to design an alternative procedure other than autogenous bone grafting. However, all known biomaterials still fall short of inducing substantial bone formation in vitro or in vivo, especially when treating large bony defects. As such there is a necessity to develop novel bone-reconstructive biomaterials that can more appropriately be utilised and can induce substantial more bone formation than current scaffolds. Using the rapid prototyping technique (Friedrich-Baur BioMed Center, Bayreuth, Germany) to develop new and improved hydroxyapatite/β-tricalcium phosphate devices, which can be predesigned to any outer shape with controlled pore structure and exhibit a unique intrinsic porosity <150µm due to the 3D-printing process to fit any skeletal bone loss site, the aim of our laboratories was to test the osteoinductive capacity of these new bioreactors in an in vitro culture system utilising adipose-derived stem cells (ADSCs). Immunofluorescent staining revealed that beside the standard surface protein expression patterns typical for ADSCs, the cells also produced osteoblast specific proteins, specifically osteocalcin, osteopontin and dentin matrix acidic phosphoprotein 1. ADSCs seeded on the surface of the biomimetic scaffolds showed constant proliferation, maintained viability and differentiation throughout the scaffold, including the small intrinsic pores. Subsequent, qRT-PCR also revealed that alkaline phosphatase and osteocalcin expression was significantly increased upon addition of osteogenic medium but even more so when human recombinant morphogenetic protein 2 (hBMP-2) was included. Immunofluorescent data of protein expression was consistent with qRT-PCR data. Taken into account with previous results by our laboratories in respect to adipose tissue as a viable inductive medium that can form substantial new bone formation in vivo the present results demonstrated that the investigated bioceramic devices possess the necessary capacity that could, together with adipose tissue, provide the next leap necessary to finally and decisively induce substantial or total new bone formation in clinical bone defects of humans

Aims

Transcription factor nuclear factor kappa B (NF-κB) plays a major role in the pathogenesis of chronic inflammatory diseases in all organ systems. Despite its importance, NF-κB targeted drug therapy to mitigate chronic inflammation has had limited success in preclinical studies. We hypothesized that sex differences affect the response to NF-κB treatment during chronic inflammation in bone. This study investigated the therapeutic effects of NF-κB decoy oligodeoxynucleotides (ODN) during chronic inflammation in male and female mice.

Fabrication of biogenic coatings with suitable mechanical properties is a key goal in orthopedics, to overcome the limitations of currently available coatings and improve the clinical results of coated implants compared to uncoated ones. In this paper, biological-like apatite coatings were deposited from a natural bone-apatite source by a pulsed electron deposition technique (PED). Bone apatite-like (BAL) films were deposited directly from bone targets, obtained by standard deproteinization of bovine tibial cortical shafts and compared to films deposited by sintered stoichiometric-hydroxyapatite targets (HA). Deposition was performed at room temperature by PED in the Ionized Jet Deposition (IJD) version. Half of the samples was annealed at 400°C for 1h (BAL_400 and HA_400). As-deposited and annealed coatings were characterized in terms of composition and crystallinity (XRD, FT-IR), microstructure and morphology (SEM-EDS, AFM) and mechanical properties (nanoindentation and micro-scratch). For the biological tests, human dental pulp stem cells (hDPSCs) were isolated from dental pulp from patients undergoing a routine tooth extraction, plated on the samples (2500 cells/cm2) and cultured for 3 weeks, when the expression of typical osteogenic markers Runx-2, osteopontin, Osx and Osteocalcin in hDPSCs was evaluated. Results showed that deposition by PED allows for a close transfer of the targets” composition. As-deposited coatings exhibited low cristallinity, that was significantly increased by post-deposition annealing, up to resembling that of biogenic apatite target. As a result of annealing, mechanical properties increased up to values comparable to those of commercial plasma-sprayed HA-coatings. In vitro biological tests indicated that BAL_400 promotes hDPSCs proliferation to a higher extent compared to non-annealed bone coating and HA-references. Furher, immunofluorescence and western blot analyses revealed that the typical osteogenic markers were expressed, indicating that BAL_400 alone can efficiently promote the osteogenic commitment of the cells, even in absence of an osteogenic medium. In conclusion, bone-like apatite coatings were deposited by PED, which closely resembled composition and structure of natural-apatite. Upon annealing at 400°C, the coatings exhibited satisfactory mechanical properties and were capable of providing a suitable microenvironment for hDPSCs adherence and proliferation and for them to reach osteogenic commitment. These results suggest that bone apatite-like thin films obtained by biogenic source may represent an innovative platform to boost bone regeneration in the orthopedic, maxillofacial and odontoiatric field

Background. Following endosteal uncemented orthopaedic device implantation, the initial implant/bone interface retains spaces and deficiencies further exacerbated by pressure necrosis and resultant bone resorption. This implant-bone space requires native bone infill through the process of de novo osteogenesis. New appositional bone formation on the implant surface is known as contact osteogenesis and is generated by osteogenic cells, including skeletal stem cells (SSCs), colonising the implant surface and depositing the extracellular bone matrix. Surface nanotopographies provide physical cues capable of triggering SSC differentiation into osteoblasts, thus inducing contact osteogenesis, translated clinically into enhanced osseointegration and attainment of secondary stability. The current study has investigated the in vitro and in vivo effects of unique nanotopographical pillar substrates on SSC phenotype and function. Methods. Adult human SSCs were immunoselected, enriched using STRO-1 antibody and cultured on control and test surfaces for 21 days in vitro. The test groups comprised Ti-coated substrates with planar or modified surfaces with nanopillar. Osteoinductive potential was analysed using qPCR and immunostaining to examine gene expression and protein synthesis. Results. Following in vitro (n=5) culture on nanopillars, the expression of osteogenic genes (ALP, Collagen 1, OPN and OCN) and of Osteopontin protein (a bone matrix protein), on Ti pillars were both significantly enhanced when compared to control or Ti planar surfaces. Conclusions. Discrete raised surface nanopillars modulate adult SSC populations in the absence of any chemical cues and enhance their osteogenic properties, an effect not observed on planar Ti constructs. Hence, these findings herald exciting opportunities to improve the implant surface design, implant osseointegration, and, ultimately, implant survival. Level of evidence. Original experimental study