Periosteum is important for bone homoeostasis through the release of bone morphogenetic proteins (BMPs) and their effect on osteoprogenitor cells. Smoking has an adverse effect on fracture healing and bone regeneration. The aim of this study was to evaluate the effect of smoking on the expression of the BMPs of human periosteum. Real-time polymerase chain reaction was performed for BMP-2,-4,-6,-7 gene expression in periosteal samples obtained from 45 fractured bones (19 smokers, 26 non-smokers) and 60 non-fractured bones (21 smokers, 39 non-smokers). A hierarchical model of BMP gene expression (BMP-2 > BMP-6 > BMP-4 > BMP-7) was demonstrated in all samples. When smokers and non-smokers were compared, a remarkable reduction in the gene expression of BMP-2, -4 and -6 was noticed in smokers. The comparison of fracture and non-fracture groups demonstrated a higher gene expression of BMP-2, -4 and -7 in the non-fracture samples. Within the subgroups (fracture and non-fracture), BMP gene expression in smokers was either lower but without statistical significance in the majority of BMPs, or similar to that in non-smokers with regard to BMP-4 in fracture and BMP-7 in non-fracture samples. In smokers, BMP gene expression of human periosteum was reduced, demonstrating the effect of smoking at the molecular level by reduction of mRNA transcription of periosteal BMPs. Among the BMPs studied, BMP-2 gene expression was significantly higher, highlighting its role in bone homoeostasis.

Aim. Osteoarthritis (OA) is caused by complex interactions between genetic and environmental factors. Epigenetic mechanisms control the expression of genes and are likely to regulate the OA transcriptome. We performed integrative genomic analyses to define methylation-gene expression relationships in osteoarthritic cartilage. Patients and Methods. Genome-wide DNA methylation profiling of articular cartilage from five patients with OA of the knee and five healthy controls was conducted using the Illumina Infinium HumanMethylation450 BeadChip (Illumina, San Diego, California). Other independent genome-wide mRNA expression profiles of articular cartilage from three patients with OA and three healthy controls were obtained from the Gene Expression Omnibus (GEO) database. Integrative pathway enrichment analysis of DNA methylation and mRNA expression profiles was performed using integrated analysis of cross-platform microarray and pathway software. Gene ontology (GO) analysis was conducted using the Database for Annotation, Visualization and Integrated Discovery (DAVID). Results. We identified 1265 differentially methylated genes, of which 145 are associated with significant changes in gene expression, such as DLX5, NCOR2 and AXIN2 (all p-values of both DNA methylation and mRNA expression < 0.05). Pathway enrichment analysis identified 26 OA-associated pathways, such as mitogen-activated protein kinase (MAPK) signalling pathway (p = 6.25 × 10-4), phosphatidylinositol (PI) signalling system (p = 4.38 × 10-3), hypoxia-inducible factor 1 (HIF-1) signalling pathway (p = 8.63 × 10-3 pantothenate and coenzyme A (CoA) biosynthesis (p = 0.017), ErbB signalling pathway (p = 0.024), inositol phosphate (IP) metabolism (p = 0.025), and calcium signalling pathway (p = 0.032). Conclusion. We identified a group of genes and biological pathwayswhich were significantly different in both DNA methylation and mRNA expression profiles between patients with OA and controls. These results may provide new clues for clarifying the mechanisms involved in the development of OA. Cite this article: A. He, Y. Ning, Y. Wen, Y. Cai, K. Xu, Y. Cai, J. Han, L. Liu, Y. Du, X. Liang, P. Li, Q. Fan, J. Hao, X. Wang, X. Guo, T. Ma, F. Zhang. Use of integrative epigenetic and mRNA expression analyses to identify significantly changed genes and functional pathways in osteoarthritic cartilage. Bone Joint Res 2018;7:343–350. DOI: 10.1302/2046-3758.75.BJR-2017-0284.R1

Aims. Exosomes (exo) are involved in the progression of osteoarthritis (OA). This study aimed to investigate the function of dysfunctional chondrocyte-derived exo (DC-exo) on OA in rats and rat macrophages. Methods. Rat-derived chondrocytes were isolated, and DCs induced with interleukin (IL)-1β were used for exo isolation. Rats with OA (n = 36) or macrophages were treated with DC-exo or phosphate-buffered saline (PBS). Macrophage polarization and autophagy, and degradation and chondrocyte activity of cartilage tissues, were examined. RNA sequencing was used to detect genes differentially expressed in DC-exo, followed by RNA pull-down and ribonucleoprotein immunoprecipitation (RIP). Long non-coding RNA osteoarthritis non-coding transcript (OANCT) and phosphoinositide-3-kinase regulatory subunit 5 (PIK3R5) were depleted in DC-exo-treated macrophages and OA rats, in order to observe macrophage polarization and cartilage degradation. The PI3K/AKT/mammalian target of rapamycin (mTOR) pathway activity in cells and tissues was measured using western blot. Results. DC-exo inhibited macrophage autophagy (p = 0.002) and promoted M1 macrophage polarization (p = 0.002). DC-exo at 20 μg/ml induced collagen degradation (p < 0.001) and inflammatory cell infiltration (p = 0.023) in rats. OANCT was elevated in DC (p < 0.001) and in cartilage tissues of OA patients (p < 0.001), and positively correlated with patients’ Kellgren-Lawrence grade (p < 0.001). PIK3R5 was increased in DC-exo-treated cartilage tissues (p < 0.001), and OANCT bound to fat mass and obesity-associated protein (FTO) (p < 0.001). FTO bound to PIK3R5 (p < 0.001) to inhibit the stability of PIK3R5 messenger RNA (mRNA) (p < 0.001) and disrupt the PI3K/AKT/mTOR pathway (p < 0.001). Conclusion. Exosomal OANCT from DC could bind to FTO protein, thereby maintaining the mRNA stability of PIK3R5, further activating the PI3K/AKT/mTOR pathway to exacerbate OA. Cite this article: Bone Joint Res 2022;11(9):652–668

Bone morphogenetic proteins (BMPs) have been widely investigated for treating non-healing fractures. They participate in bone reconstruction by inducing osteoblast differentiation, and osteoid matrix production. 1. The human recombinant protein of BMP-7 was among the first growth factors approved for clinical use. Despite achieving comparable results to autologous bone grafting, severe side effects have been associated with its use. 2. Furthermore, BMP-7 was removed from the market. 3. These complications are related to the high doses used (1.5-40 miligrams per surgery). 2. compared to the physiological concentration of BMP in fracture healing (in the nanogram to picogram per milliliter range). 4. In this study, we use transcript therapy to deliver chemically modified mRNA (cmRNA) encoding BMP-7. Compared to direct use of proteins, transcript therapy allows the sustained synthesis of proteins with native conformation and true post-translational modifications using doses comparable to the physiological ones. 5. Moreover, cmRNA technology overcomes the safety and affordability limitations of standard gene therapy i.e. pDNA. 6. BMP-7 cmRNA was delivered using Lipofectamine™ MessengerMAX™ to human mesenchymal stromal cells (hMSCs). We assessed protein expression and osteogenic capacity of hMSCs in monolayer culture and in a house-made, collagen hydroxyapatite scaffold. Using fluorescently-labelled cmRNA we observed an even distribution after loading complexes into the scaffold and a complete release after 3 days. For both monolayer and 3D culture, BMP-7 production peaked at 24 hours post-transfection, however cells transfected in scaffolds showed a sustained expression. BMP-7 transfected hMSCs yielded significantly higher ALP activity and Alizarin red staining at later timepoints compared to the untransfected group. Interestingly, BMP-7 cmRNA treatment triggered expression of osteogenic genes like OSX, RUNX-2 and OPN, which was also reflected in immunostainings. This work highlights the relevance of cmRNA technology that may overcome the shortcomings of protein delivery while circumventing issues of traditional pDNA-based gene therapy for bone regeneration. Acknowledgement: This work has been performed as part of the cmRNAbone project and has received funding from the European Union's Horizon 2020 research and innovation programme under the Grant Agreement No 874790

Lubricin is a proteoglycan that is a boundary lubricant in synovial joints and both a surface and collagen inter-fascicular lubricant in ligaments. The purpose of this study was to characterise the mRNA levels for lubricin in the anterior cruciate ligament (ACL), posterior cruciate ligament (PCL), medial collateral ligament (MCL), and lateral collateral ligament (LCL) in aging and surgically-induced menopausal rabbits. We hypothesised that lubricin mRNA levels would be increased in ligaments from aging and menopausal rabbits compared with ligaments from normal rabbits. All four knee ligaments (ACL, PCL, MCL, LCL) were isolated from normal (1-year-old rabbits, n=8), aging (3-year-old rabbits, n=6), and menopausal (1-year-old rabbits fourteen weeks after surgical ovariohysterectomy, n=8) female New Zealand White rabbits. RT-qPCR was used to evaluate the mRNA levels for lubricin normalised to the housekeeping gene 18S. After removing outliers, data for normal, aging, and menopausal rabbits for each knee ligament (ACL, PCL, MCL, LCL) were compared using ANOVA with linear contrasts or Kruskal-Wallis test with Conover post-hoc analysis. For ACLs, the mRNA levels for lubricin were increased in menopausal and aging rabbits compared with normal rabbits (p<0.056). For PCLs, trends for increased lubricin mRNA levels were found when comparing menopausal and aging rabbits with normal rabbits (p<0.092). For MCLs, the mRNA levels for lubricin were increased in menopausal and aging rabbits compared with normal rabbits (p<0.050). For LCLs, no differences in lubricin mRNA levels were detected comparing the three groups. For all four knee ligaments (ACL, PCL, MCL, LCL), no differences in lubricin mRNA levels were detected comparing the ligaments from menopausal rabbits with those from aging rabbits. Lubricin plays a role in collagen fascicle lubrication in ligaments (1,2). Increased lubricin gene expression was associated with mechanical changes (including decreased modulus and increased failure strain) in the aging rabbit MCL (3). Detection of similar molecular changes in the ACL, and possibly the PCL, may indicate that their mechanical properties may also change as a result of increased lubricin gene expression, thereby potentially pre-disposing these ligaments to damage accumulation. Compared to aging ligaments, aging tendons exhibited decreased lubricin gene and protein expression, and increased stiffness (4). Although opposite changes than aging ligaments, these findings support the relationship between lubricin and modulus/stiffness. The similarities between ligaments in the aging and menopausal groups may suggest that surgically-induced menopause results in a form of accelerated aging in the rabbit ACL, MCL and possibly PCL

Tissue regeneration using growth factors has disadvantages while needing to use supraphysiological growth factor concentrations. Gene therapy has been proposed as alternative. Unfortunately, drawbacks such as the use of viruses and the inefficiency of non-viral systems jeopardize clinical translation. mRNA-based transcript therapy is a novel approach that may solve plasmid DNA-based gene therapy limitations. mRNA molecules can be chemically modified in order to improve stability and immunogenicity. Chemically modified mRNA (cmRNA) is much more efficient than pDNA in delivering genes into the cell. The combination of biomaterials with cmRNA is interesting for the tissue engineering and regenerative medicine field. The resulting construct, known as Transcript-Activated Matrix, may act as a cmRNA delivery platform while supporting cell proliferation, extracellular matrix deposition and ultimately de novo tissue formation. Our work and the work of others demonstrated that the use of Transcript-Activated Matrix prolonged transgene expression and enhanced protein translation. This presentation will provide an overview of ongoing research from our group on cmRNA for improving bone repair with a particular focus on Transcript-Activated Matrix for enhancing osteogenesis. Results of our investigation in vitro with stem cells, ex vivo using tissue culture and in vivo using rat models will be presented

Introduction: Periosteum is a tissue with pluripotential mesenchymal cells (MSCs). During fracture repair several growth factors are released from periosteum, including bone morphogenetic proteins (BMPs), which induce the differentiation of bone marrow stromal cells towards the osteoblastic lineage, therefore increasing the pool of mature bone forming cells and enhance the differentiated function of osteoblasts. The purpose of our study is to evaluate the expression of periosteal BMPs mRNA from fracture samples, collected within 24 hours of fracture and to compare it with BMPs expression from periosteal samples of normal (non-fractured) bones. Materials and Methods: Periosteum samples were collected from 25 patients with recent fracture (during the past 24 hours) (age: 12–80) and 25 individuals without fracture (age: 10–73). BMPs (BMP2, BMP4, BMP6) mRNA levels were analysed by Real Time RT-PCR by using the Light Cycler machine and PBGD as a housekeeping gene. Results: BMP2 mRNA levels were significantly higher (p< 0.05) in normal samples (median:12.15) than in fracture (median:4.39). BMP6 and BMP4 mRNA expression followed similar pattern to that of BMP2 but in significant lower levels. In normal samples, BMP4 mRNA median levels were 1.99, while in fracture samples the levels were significantly lower (median:0.35), (p< 0.05). BMP6 mRNA levels were also higher in normal samples (median:2.21) than in fractures (median:1.87) (p> 0.05). Furthermore, the decrease of BMPs mRNA levels in fracture samples was higher for BMP4 followed by BMP2 and BMP6. Discussion: Our results indicate high BMP2 mRNA levels expressed from periosteal cells. In recent fractures there is a significant reduction of BMP2 compared to normal samples; however, the expression of BMP2 remains more elevated in comparison to the other BMPs highlighting the potential role of BMP2 at the initiation of healing process of fractures. BMP6 and BMP4 expression was similar among normal periosteal cells while levels of BMP6 were higher than BMP4 in fracture periosteal cells. The suppression of BMP6 expression was minimum and less significant than BMP2 and BMP4 suppression indicating the potential role of BMP6 at the early stages of MSCs differentiation in periosteum. On the other hand, BMP4 remains in low levels in any confrontation and seems that plays a minor role in early healing process of fracture. BMPs are considered to play central role in fracture response and bone remodelling but further investigation has to be done as much in their correlation and toward other growth factors as in their expression levels during bone fracture repair process

Our aim was to determine the effect of denervation on repair-associated mRNA levels in the MCL after partial tear. Cohorts of rabbits underwent partial MCL tear with or without concomitant femoral nerve transection. Ligaments were harvested, RNA extracted and RT-PCR was performed using rabbit-specific primers for repair-associated molecules at three days, two wks, six wks and sixteen wks post-injury. Angiogenesis genes MMP3, MMP13, matrix components Collagen I and III and growth factors TGF-ß and NGF mRNA levels were increased in the denervated group at two-weeks post-injury (p< 0.05). Denervation significantly alters mRNA levels during the early stages of rabbit MCL healing. To determine the effect of denervation on repair-associated mRNA levels in the injured medial collateral ligament (MCL). Previous experiments revealed that denervation impairs healing of the MCL. We hypothesized that denervation would decrease repair-associated mRNA levels in the injured MCL when compared with normally innervated injured MCL. Adult, skeletally mature female rabbits were assigned to one of four groups: unoperated control, femoral nerve transection alone (denervated controls), MCL partial tear and denervated MCL partial tear. At three days, two weeks, six weeks or sixteen weeks post-surgery, cohorts of six rabbits from each experimental group were killed. Control rabbits were assessed at two weeks. Ligaments were harvested, RNA extracted and RT-PCR was performed using rabbit-specific primers. In the denervated injury group, mRNA levels of angiogenesis genes MMP-3 and MMP-13, matrix components Collagen I and III and growth factors TGF-ß and NGF had all increased at two-weeks post-injury, in comparison to non-denervated (p< 0.05). We also found increased levels of MMP-3 and NGF mRNA in the denervated group at sixteen weeks post injury (p< 0.05). The mRNA levels of the housekeeping gene GAPDH were increased in the denervated group only at three days post injury (p< 0.05). Of note, TGF-ß mRNA levels were significantly decreased in the denervated group at three days post injury (p< 0.05). Contrary to our initial hypothesis, denervation increases mRNA levels for many important molecules during the early stages of MCL healing. Additional research will be required to explain how and why denervation impairs ligament healing. No previous study has shown that innervation regulates mRNA levels in healing ligament

Cytokine mediated activation of osteoclasts can lead to peri-implant osteolysis and aseptic loosening. The aim of this study was to determine the IL-1β and TNFα mRNA cytokine expression profile of human macrophages when stimulated with polyethylene particles using relative quantitative real-time polymerase chain reaction (rqRT-PCR). Human peripheral blood monocytes or human monocytes from the cell line THP-1 were used in this study. rqRT-PCR conditions were optimized by stimulating human macrophages with 200ng/ml lipopolysaccharide (LPS). The median CV% value for duplicate measures was 12.6 (range 4.5–54). Stimulation assays were performed using unfractionated endotoxin-free commercial polyethylene particles (median size 7μm); or fractionated particles (size range 0.1–1.2μm). Human macrophages were stimulated with high dose unfractionated polyethylene particles at 0, 3500 or 10500 mm. 3. /cell or with fractionated polyethylene particles at 0 and 100mm. 3. /cell at time points 0 and 3 hours. Low dose unfractionated polyethylene stimulation was performed on THP-1 cells at 0, 50, 100, 1000 and 10000 mm. 3. /cell. In all experiments LPS stimulation was used as a positive control. RNA was extracted and rqRT-PCR was performed using standard techniques. High dose unfractionated polyethylene stimulation did not result in a significant difference in cytokine mRNA levels between groups. Using fractionated polyethylene, a small increase in IL-1β mRNA was identified (21% versus maximal stimulation using LPS). Low dose unfractionated polyethylene stimulation of THP-1 cells demonstrated dose dependent decreases in TNFα and IL-1β mRNA expression that was not due to inhibition of RNA extraction or a decrease of cell viability. Endotoxin-free polyethylene particles do not appear to be a major stimulus for IL-1β and TNFα mRNA production as measured by rqRT-PCR. We did observe a small positive effect on IL-1β mRNA expression using a fractionated polyethylene stimulus. However it remains unclear whether this effect is due to fractionation of particles into the submicron range or is due to introduction of endotoxin during the filtration process

Methods: Sixty pseudo-capsular tissues from loose shoulder, hip and knee (20 each) arthroplasties and 30 capsular tissues from primary total joint arthroplasty (TJA) patients (10 each; 12 rheumatoid arthritis [RA], 18 osteo-arthrosis [OA]) were investigated for mRNA and protein expressions of IL-1ß (interleukin-1 b, IL-1Ra (interleukin-1 receptor antagonist), MMP 1 (matrix matalloproteinase-1)-, TIMP 2 (tissue inhibitor of MMPs-2) using in situ hybridisation and immunohistochemistry. Polyethylene and metal debris in the same sections were semi-quantified simultaneously. Results: IL-1ß mRNA and proteins were expressed in most RA primary and revision tissues and were less expressed in OA primary tissues. In contrast, IL-1Ra mRNA was found in most primary OA tissues and less in RA primary and the revision tissues. The ratio of staining intensities of IL-1ß/IL-1Ra mRNA was higher in revision and primary RA tissues compared with the primary OA tissues. MMP-1 protein expression was correlated with the IL-1ß/IL-1Ra ratio. Polyethylene (PE) debris was found in 56 out of 60 of the revision tissues. Their sizes were different in the hip (mainly small, < 30 mm in diameter), the knee (mainly large, > 300 mm) and the shoulder (all sizes). The expressions of the detected factors were highly correlated with the concentration of the PE debris but with not their sizes. Conclusions: The high ratio of IL-1ß/IL-1Ra in primary RA and revision tissues and its correlation with MMP-1 expression and PE debris concentration indicated that an over-expression of IL-1ß and/or regulation downwards of IL-1Ra is an important event in inflammatory disorders and the foreign body reaction in TJA. A therapeutic strategy with IL-1Ra, that has been considered in RA treatment may thus contribute to the longevity of prosthesis of a TJA

Periosteum is a specialized connective tissue that surrounds bone, containing progenitor cells that develop into osteoblasts. The osteo-progenitor cells along with growth factors, such as BMPs, play critical role in development, reconstruction and bone formation. Aim: to evaluate the expression of BMPs in human periosteum and in different subrgroups, including different donor sites, gender, and smoking habits. Gene expression of BMPs 2,4,6,7 was performed in 60 periosteal samples using quantitative RT-PCR. Samples were obtained from 32 men/28 women, 22 smokers/38 non-smokers, 29 lower/31 upper extremities. BMP2 gene expression was significantly higher (median: 12.02, p< 0.05) than the mRNA levels of BMPs 4,6,7 (median: 1.36, 2.55, 0.04) in all samples. BMP2 mRNA levels were higher in large compared to small bones (median: 13.4 vs 9.48), while BMPs 4,6,7 gene expression was similar (1.3 vs 1.4, 2.7 vs 2.1, 0.04 vs 0.03, respectively). In lower extremities, BMPs mRNA levels were higher than in the upper; the same was detected in non-smokers versus smokers group (BMPs2,4,6,7: 13.9 vs 1.5, 3.1 vs 0.048, 8.7 vs 1.06, 1.6 vs 0.026, respectively). mRNA transcripts of BMP2 were higher in men than women (median: 13.1 vs 10.8). In our study, BMP2 expression is characteristically higher than that of BMP4, BMP6 and BMP7, highlighting the critical role that BMP2 plays in bone homeostasis. Furthermore, the elevated expression of BMP2 in men towards women, and of all BMPs of the lower extremity samples indicate the effect of hormones and mechanical factors in periosteal BMPs gene regulation; while the effect of smoking is reflected in the reduction of BMPs expression in smokers

Phagocytosis of wear particles by perimplant macrophages results in cytokine release and osteoclast activation and osteolysis. Some investigators have proposed that this response may be mediated by adherent endotoxin. The aim of this study was to determine the role of endotoxin in modulating pro-inflammatory cytokine mRNA expression of macrophages when stimulated with titanium particles using relative quantitative real-time polymerase chain reaction (rqRT-PCR). Human peripheral blood mononuclear cells were isolated from healthy subjects and plated in chamber slides. Three types of titanium particles were prepared; commercially pure titanium particles (cpTi), endotoxin stripped particles and endotoxin stripped particles with endotoxin (LPS) added back. Endotoxin levels of 450, 0 and 140 Eu/ml respectively were confirmed by high sensitivity Limulus Amebocyte Lysate assay. Macrophages were stimulated with particle concentrations of 0, 8.3, 83 and 830 particles per cell at time points 0 and 3 hours. LPS (200ng/ml) was used as a positive control. rqRT-PCR was performed using standard techniques. Stimulation of human macrophages with cpTi demonstrated a significant dose dependent increase in TNFα, IL-1A, IL-1B and, IL-6. (Kruskal-Wallis p=0.01, p=0.017, p=0.001 and p=0.013 respectively). IL-18 mRNA levels were not increased (P> 0.05). The expression of mRNA following stimulation with the highest dose of titanium particles was similar to that following LPS stimulation. Endotoxin-free cpTi particles did not elicit any increase in mRNA expression above base line levels (P > 0.05, all cytokines). This lack of response was rescued in endotoxin-stripped particles with LPS added back. Particle dose dependent increases in cytokine mRNA levels were observed for TNFα, IL-1A, IL-1B and, IL-6 mRNA but not IL-18 (p=0.01, p=0.01, p=0.01, p=0.05 and p=0.> 0.05 respectively). Our results show that adherent endotoxin plays a role in modulating particle induced pro-inflammatory cytokine mRNA expression in-vitro. Further study is required in evaluating the role of adherent endotoxin in vivo

Purpose: Vascular Endothelial Growth Factor (VEGF) plays an important role in promoting angiogenesis and osteogenesis during fracture repair. Our previous studies have shown that cell-based VEGF gene therapy accelerates bone healing of a rabbit tibia segmental bone defect in-vivo, and increases osteoblast proliferation and mineralization in-vitro. The aim of this project was to examine the effect of exogenous human VEGF (hVEGF) on the endogenous rat VEGF messenger RNA (mRNA) expression in a cell-based gene transfer model. Method: The osteoblasts were obtained from the rat periosteum. The fibroblasts were obtained from the rat dermal tissue. The cells were then cultured to reach 60% confluence and transfected with hVEGF using Superfect. Four groups were:. osteoblast-hVEGF,. fibroblast-hVEGF,. Osteoblasts alone, and. Fibroblasts only. The cultured cells were harvested at 1, 3 and 7 days after the transfection. The total mRNA was extracted (TRIZOL); both hVEGF and rat VEGF mRNA were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and quantified by VisionWorksLS. Results: The hVEGF mRNA was detected by RT-PCR from transfected osteoblasts after three days of gene transfection. The hVEGF mRNA expression in transfected fibroblasts increased exponentially at days 1, 3 and 7 after the transfection. We compared the endogenous rat VEGF mRNA expression level of the osteoblasts or fibroblasts that were transfected with hVEGF with the cells without the transfection. The hVEGF transfected osteoblasts had a greater rat VEGF mRNA expression than the non-transfected osteoblasts. Furthermore, when hVEGF was transfected to the rat fibroblasts, the endogenous mRNA expression level measured was also greater than that from the non-transfected fibroblasts. Rat VEGF mRNA expression increased in the first three days of the hVEGF transfection, but the expression level was reduced at Day 7. Conclusion: These results suggest that cell-based hVEGF gene therapy enhances endogenous rat VEGF mRNA expression in both osteoblasts and fibroblasts

Aims. This study aimed to determine the expression and clinical significance of a cartilage protein, cartilage oligomeric matrix protein (COMP), in knee osteoarthritis (OA) patients. Methods. A total of 270 knee OA patients and 93 healthy controls were recruited. COMP messenger RNA (mRNA) and protein levels in serum, synovial fluid, synovial tissue, and fibroblast-like synoviocytes (FLSs) of knee OA patients were determined using enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and immunohistochemistry. Results. COMP protein levels were significantly elevated in serum and synovial fluid of knee OA patients, especially those in the advanced stages of the disease. Serum COMP was significantly correlated with radiological severity as well as measures of body composition, physical performance, knee pain, and disability. Receiver operating characteristic curve analysis unveiled a diagnostic value of serum COMP as a biomarker of knee OA (41.64 ng/ml, area under the curve (AUC) = 1.00), with a sensitivity of 99.6% and a specificity of 100.0%. Further analysis uncovered that COMP mRNA expression was markedly upregulated in the inflamed synovium of knee OA, consistent with immunohistochemical staining revealing localization of COMP protein in the lining and sub-lining layers of knee OA inflamed synovium. Most notably, relative COMP mRNA expression in knee OA synovium was positively associated with its protein levels in serum and synovial fluid of knee OA patients. In human knee OA FLSs activated with tumour necrosis factor-alpha, COMP mRNA expression was considerably up-regulated in a time-dependent manner. Conclusion. All results indicate that COMP might serve as a supportive diagnostic marker for knee OA in conjunction with the standard diagnostic methods. Cite this article: Bone Joint Res 2024;13(6):261–271

Aims. Lumbar spinal stenosis (LSS) is a common skeletal system disease that has been partly attributed to genetic variation. However, the correlation between genetic variation and pathological changes in LSS is insufficient, and it is difficult to provide a reference for the early diagnosis and treatment of the disease. Methods. We conducted a transcriptome-wide association study (TWAS) of spinal canal stenosis by integrating genome-wide association study summary statistics (including 661 cases and 178,065 controls) derived from Biobank Japan, and pre-computed gene expression weights of skeletal muscle and whole blood implemented in FUSION software. To verify the TWAS results, the candidate genes were furthered compared with messenger RNA (mRNA) expression profiles of LSS to screen for common genes. Finally, Metascape software was used to perform enrichment analysis of the candidate genes and common genes. Results. TWAS identified 295 genes with permutation p-values < 0.05 for skeletal muscle and 79 genes associated for the whole blood, such as RCHY1 (PTWAS = 0.001). Those genes were enriched in 112 gene ontology (GO) terms and five Kyoto Encyclopedia of Genes and Genomes pathways, such as ‘chemical carcinogenesis - reactive oxygen species’ (LogP value = −2.139). Further comparing the TWAS significant genes with the differentially expressed genes identified by mRNA expression profiles of LSS found 18 overlapped genes, such as interleukin 15 receptor subunit alpha (IL15RA) (PTWAS = 0.040, PmRNA = 0.010). Moreover, 71 common GO terms were detected for the enrichment results of TWAS and mRNA expression profiles, such as negative regulation of cell differentiation (LogP value = −2.811). Conclusion. This study revealed the genetic mechanism behind the pathological changes in LSS, and may provide novel insights for the early diagnosis and intervention of LSS. Cite this article: Bone Joint Res 2023;12(6):387–396

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

Purpose. Angiogenesis and osteogenesis are essential for bone growth, fracture repair, and bone remodeling. VEGF has an important role in bone repair by promoting angiogenesis and osteogenesis. In our previous study, endothelial progenitor cells (EPCs) promoted bone healing in a rat segmental bone defect as confirmed by radiological, histological and microCT evaluations (Atesok, Li, Schemitsch 2010); EPC treatment of fractures resulted in a significantly higher strength by biomechanical examination (Li, Schemitsch 2010). In addition, cell-based VEGF gene transfer has been effective in the treatment of segmental bone defects in a rabbit model (Li, Schemitsch et al 2009); Purpose of this study: Evaluation of VEGF gene expression after EPC local therapy for a rat segmental bone defect. Method. Rat bone marrow-derived EPCs were isolated from the rat bone marrow by the Ficoll-paque gradient centrifuge technique. The EPCs were cultured for 7 to 10 days in endothelial cell growth medium with supplements (EGM-2-MV-SingleQuots, Clonetics). and collected for treatment of the rat segmental bone defect. EPCs were identified by immunocytochemistry staining with primary antibodies for CD34, CD133, FLK-1, and vWF. A total of fifty six rats were studied. A five millimeter segmental bone defect was created in the middle 1/3 of each femur followed by mini plate fixation. The treatment group received 1×106 EPCs locally at the bone defect and control animals received saline only. Seven control and seven EPC treated rats were included in each group at 1, 2, 3 and 10 weeks. Animals were sacrificed at the end of the treatment period, and specimens from the fracture gap area were collected and immediately frozen. Rat VEGF mRNA was measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and quantified by VisionWorksLS. All measurements were performed in triplicate. Results. Cultured EPCs at 1 week showed positive staining for CD34, CD133, Flk-1 and vWf markers. The EPC group had a greater VEGF expression than the control group at week 1, 2 and 3 but not at week 10. Three VEGF isoforms were detected in this rat model: VEGF120, VEGF164 and VEGF188. VEGF120 and VEGF164 levels peaked at two weeks, while VEGF188 levels peaked at three weeks. All three VEGF isoform levels were low at ten weeks. Conclusion. EPC-based therapy for a segmental bone defect results in increased VEGF expression during the early period of fracture repair. In addition, the specific VEGF isoform may be a key regulator of the bone healing process. These findings demonstrate that EPCs promote fracture healing by increasing VEGF levels and thus stimulating angiogenesis, a process that is essential for early callus formation and bone regeneration

Aims: Previous work at this institution has demonstrated that perfusion beneath circumferential negative pressure wound therapy (NPWT) is decreased, which conflicts with most studies on NPWT and perfusion. This study investigates perfusion beneath non-circumferential NPWT in humans and also discusses the potential methodology flaw common to all previous research in this field.

Methods: Tests were conducted on both lower limbs of six volunteers (N=12). Volunteers were sequentially randomised into two groups, which would receive different suction pressures (−400 mmHg and −125 mmHg). A doughnut shaped NPWT dressing was placed over the shin of each leg. The central hole allowed for measurement of the transcutaneous partial pressure of oxygen (tcpO2), an indirect measure of perfusion. Readings were taken every five minutes throughout the experiment. After acquiring readings for 15 minutes to establish a baseline, suction was switched on and readings were taken for another 15 minutes. Suction was then disconnected and readings were taken for 15 minutes. Suction was then reapplied and the sequence was repeated. Data were analysed using the Wilcoxon and Mann-Whitney tests.

Results: On applying suction pressures of −400 mmHg, there was a significant reduction of the tcpO2 (mean reduction 7.35 mmHg, SD 7.4, p< 0.0005). At −125 mmHg, there was also a significant reduction of the tcpO2 (mean reduction 5.10 mmHg, SD 7.4, p< 0.0005). Although there was a tendency for greater reductions in the −400 mmHg group this was not significantly different to the −125 mmHg group (p=0.07).

Conclusion: NPWT reduces tissue perfusion, with higher suction pressure resulting in greater reductions in perfusion. Studies on perfusion using laser Doppler, which report findings contrary to these, may be flawed due to the measuring technique of this device. The compressive forces of NPWT are likely to result in false increased readings on application of suction when using the laser Doppler.

This represents a paradigm shift in our understanding of NWPT and that these dressings should be used with caution on tissues with compromised perfusion.

Aims

Impaired fracture repair in patients with type 2 diabetes mellitus (T2DM) is not fully understood. In this study, we aimed to characterize the local changes in gene expression (GE) associated with diabetic fracture. We used an unbiased approach to compare GE in the fracture callus of Zucker diabetic fatty (ZDF) rats relative to wild-type (WT) littermates at three weeks following femoral osteotomy.

Aims

Tissue inhibitors of metalloproteinases (TIMPs) are the endogenous inhibitors of the zinc-dependent matrix metalloproteinases (MMP) and A disintegrin and metalloproteinases (ADAM) involved in extracellular matrix modulation. The present study aims to develop the TIMPs as biologics for osteoclast-related disorders.