Fracture non-union can be as high as 20% in certain clinical scenarios and has a high associated socioeconomic burden. Boron has been shown to regulate the Wnt/β-catenin pathway in other bodily processes. However, this pathway is also critical for bone healing. Here we aim to demonstrate that the local delivery of boric acid can accelerate bone healing, as well as to elucidate how boric acid, via the regulationtheWnt/β-catenin pathway, impacts theosteogenic response of bone-derived osteoclasts and osteoblasts during each phase of bone repair. Bilateral femoral cortical defects were created in 32 skeletally mature C57 mice. On the experimental side, boric acid (8mg/kg concentration) was injected locally at the defect site whereas on the control side, saline was used. Mice were euthanized at 7, 14, and 28 days. MicroCT was used to quantify bone regeneration at the defect. Histological staining for ALP and TRAP was used to quantify osteoblast and osteoclast activity respectively. Immunohistochemical antibodies, β-catenin and CD34 were used to quantify active β-catenin levels and
INTRODUCTION. Stimulation of
Osteosarcoma is the most common primary bone tumour worldwide. This disease presents a formidable challenge to the orthopaedic surgeon, with a mortality rate of 30 per cent, even after surgical clearance. Aberrant Wnt signalling has been implicated in the pathogenesis osteoblastic tumours. The objective of this study is 2 fold- to investigate if osteosarcoma does indeed demonstrate aberrant Wnt signaling, and if so, does osteosarcoma respond to a novel Wnt inhibitor(ETC159). This can potentially lead to the development of a new adjuvant treatment modality for osteosarcoma. A novel Wnt signaling pathway protein antibody (YJ5) was used in immunihistochemistry staining of clinical osteosarcoma samples. A Wnt high osteosarcoma cell line(SJSA-1) was then implanted subcutaneously in a mouse model. These mice were treated with a novel PORCN inhibitor, ETC 159 for a period of 4 weeks in a two-arm randomised control study. The results of treatment were evaulated by clinical outcome parameters as well as immunohstochemistry. 100 per cent of clinical osteosarcoma samples demonstrated increased WLS expression and Wnt protein expression. SJSA-1 showed no significant decrease in tumour volume after 30 days of drug treatment (3070 SD 625 mm3 vs 3480 SD 433 mm3 p= 0.605 and 2060 SD 209 vs 1677 SD 213 mm3 p=0.219 respectively). Significantly, SJSA-1 demonstrated increased tumour necrosis in the treatment arm(30–60 percent increase across all samples p < 0 .005) Treated tumours also demonstrated markedly less
Introduction. Osteoarthritis (OA) has historically been thought of as a degenerative joint disease, but inflammation and
Tenomodulin (Tnmd) is the best known mature tendon factor for tendon and ligament tissues with reported important regulatory roles1. In addition, Tnmd C-terminal cysteine-rich domain has been descibed to exert anti-angiogenic functions in in vitro angiogenic assays as well as in vivo models of tendon injury and age-associated cardiac valve diseases1, 2. Interestingly, Tnmd expresson in the intervertebral disc (IVD), which is normally avascular tissue, has been also suggested3. Hence, the purpose of this study was first, to map the exact expression pattern of Tnmd during IVD development and aging and second, by implementing Tnmd-knockout mouse model, to examine if Tnmd plays a role in IVD homeostasis. Histological analyses (hematoxylin/eosin, Safranin O, CD31 for endothelium, TUNEL for apoptosis and type X collagen and Runx2 for hypetrophy) were performed on Tnmd −/−, Tnmd −/− and chondromodulin I Chmd 1 −/− (Tnmd only homolog) double knockout and wild type mice WT (n = three to five) to examine IVD degeneration. Real time PCR was implemented to explore gene expression chnages in annulus fibrous (AF) between Tnmd −/− and WT mice. In addition, outer AF (OAF) cells were isolated from both genotypes to further determine cellular phenotype and assess effects on co-culture with human umbical vein endothelial cells (HUVECs). Statistical differences between two groups were determined with t-test. In multiple comparisons, one-way ANOVA was followed by Bonferroni post-hoc correction. Tnmd was expressed in a temporal manner in OAF and to very low extent in NP. Tnmd −/− mice exhibited more rapid progression of age-related IVD degeneration. These signs included smaller collagen fibril diameter, reduced multiple IVD- and tendon/ligament-related gene expression, induced
Surgical failure, mainly caused by loosening implants, causes great mental and physical trauma to patients. Improving the physicochemical properties of implants to achieve favourable osseointegration will continue to be the focus of future research. Strontium (Sr), a trace element, is often incorporated into hydroxyapatite (HA) to improve its osteogenic activity. Our previous studies have shown that miR-21 can promote the osteogenic differentiation of mesenchymal stem cells by the PI3K/β-catenin pathway. The aim of this study is to fabricate a SrHA and miR-21 composite coating and it is expected to have a favorable bone healing capability. Ti discs (20 mm diameter and one mm thickness for the in vitro section) and rods (four mm diameter and seven mm length for the in vivo section) were prepared by machining pure Ti. The Ti cylinders were placed in a Teflon-lined stainless-steel autoclave for treating at 150°C for 24 h to form SrHA layer. The miR-21 was encapsulated in nanocapsules. The miR-21 nanocapsules were mixed with CMCS powder to form a gel-like sample and uniformly coated on the SrHA modifed Ti. Osteoblast-like MG63 cells were cultured on SrHA and miR-21 modified Ti, Cell proliferation activity and osteogenesis-related gene expression were evaluated. A bone defect model was established with mature New Zealand to evaluate the osseointegration. Cylindrical holes (four mm in diameter) were created at the distal femur and tibial plateau. Each rabbit was implanted with four of the aforementioned rods (distal femur and tibial plateau of the hind legs). After implantation for one, two and three months, the rabbits were observed by X-ray and scanned using u-CT. Histological and Immunohistochemical analysis were performed to examine the osteogenic markers. A biomechanical push-in test was used to assess the bone-implant bonding strength. Both SrHA nanoparticles with good superhydrophilicity and miR-21 nanocapsules with uniform sizes were distributed evenly on the surface of the Ti. In vitro experiments revealed that the composite coating was beneficial to osteoblast proliferation, differentiation and mineralization. In vivo evaluations demonstrated that this coating could not only promote the expression of angiogenic factor CD31 but also enhance the expression of osteoblastic genes to facilitate angio-osteogenesis. In addition, the composite coating also showed a decreased RANKL expression compared with the miR-21 coating. As a result, the SrHA/miR-21 composite coating promoted new bone formation and mineralization and thus enhanced osseointegration and bone-implant bonding strength. A homogeneous SrHA and miR-21 composite coating was fabricated by generating pure Ti through a hydrothermal process, followed by adhering miR-21 nanocapsules. This coating combined the favorable physicochemical properties of SrHA and miR-21 that synergistically promoted
The treatment of critical-sized bone defects still remains today a challenge, especially when the surrounding soft, vascularized and innervated tissues have been damaged - a lack of revascularization within the injured site leading to physiological disorders, from delayed healing to osteonecrosis. The axial insertion of a vascular bundle (e.g. arterio-venous loop, AVL) within a synthetic bone filler to initiate and promote its revascularization has been foreseen as a promising alternative to the current strategies (e.g., vascularized free flaps) for the regeneration of large bone defects. In a previous work, we showed that the insertion of a vein in a 3D-printed monetite scaffold induced its higher revascularization than AVL, thus a possible simplification of the surgical procedures (no microsurgery required). Going further, we investigate in this study whether or not the presence of a vein could stimulate the formation of mineralized tissue insides a synthetic scaffold filled with bone marrow and implanted in ectopic site. Monetite scaffolds were produced by additive manufacturing according to a reactive 3D-printing technique co-developed by the authors then thoroughly characterized. Animal study was performed on 14 male Wistar rats. After anesthesia and analgesia, a skin medial incision in rat thigh allowed the site on implantation to be exposed. Bone marrow was collected on the opposite femur through a minimally invasive procedure and the implant was soaked with it. For the control group (N=7), the implant was inserted in the incision and the wound was closed whereas the femoral bundle was dissected and the vein inserted in the implant for the experimental group (N=7). After 8 weeks animals were sacrificed, the implant collected and fixed in a 4% paraformaldehyde solution. Explants were characterized by µCT then embedded in poly-methyl methacrylate prior SEM, histology and immunohistochemistry. Images were analyzed with CT-Analyzer (Bruker) and ImageJ (NIH) and statistical analyses were carried out using SPSS (IBM). Implants were successfully 3D-printed with a +150 µm deviation from the initial CAD. As expected, implants were composed of 63%wt monetite and 37%wt unreacted TCP, with a total porosity of 44%. Data suggested that scaffold biodegradation was significantly higher when perfused by a vein. Moreover, the latter allowed for the development of a dense vascular network within the implant, which is far more advanced than for the control group. Finally, although mineralized tissues were observed both inside and outside the implant for both groups, bone formation appeared to be much more important in the experimental one. The ectopic formation of a new mineralized tissue within a monetite implant soaked with bone marrow seems to be highly stimulated by the simple presence of a vein alone. Although AVL have been studied extensively, little is known about the couple
The Masquelet or induced membrane technique (IMT) is a two-stage surgical procedure used for the treatment of segmental bone defects. In this technique, the defect is first filled with a polymethyl methacrylate (PMMA) spacer, which triggers the formation of a membrane that will encapsulate the defect. During the second surgery, the spacer is carefully removed and replaced by autologous bone graft while preserving the membrane. This membrane is vascularized, contains growth factors, and provides mechanical stability to the graft, all of which are assumed to prevent graft resorption and promote bone healing. The technique is gaining in popularity and several variations have been introduced in the clinical practice. For instance, orthopaedic surgeons now often include antibiotics in the spacer to treat or prevent infection. However, the consequences of this approach on the properties of the induce membrane are not fully understood. Accordingly, in a small animal model, this study aimed to determine the impact on the induced membrane of impregnating spacers with antibiotics frequently used in the IMT. We surgically created a five-mm segmental defect in the right femur of 25 adult male Sprague Dawley rats. The bone was stabilized with a plate and screws before filling the defect with a PMMA spacer. Animals were divided into five equal groups according to the type and dose of antibiotics impregnated in the spacer: A) no antibiotic (control), B) low-dose tobramycin (1.2 g/40 g of PMMA), C) low-dose vancomycin (1 g/40 g of PMMA), D) high-dose tobramycin (3.6 g/40 g of PMMA), E) high-dose vancomycin (3 g/40 g of PMMA). The animals were euthanized three weeks after surgery and the induced membranes were collected and divided for analysis. We assessed the expression of selected genes (Alpl, Ctgf, Runx2, Tgfb1, Vegfa) within the membrane by quantitative real-time PCR. Moreover, frozen sections of the specimens were used to quantify vascularity by immunohistochemistry (CD31 antigen), proliferative cells by immunofluorescence (Ki-67 antigen), and membrane thickness. Microscopic images of the entire tissue sections were taken and analyzed using FIJI software. Finally, we measured the concentration of vascular endothelial growth factor (VEGF) in the membranes by ELISA. No significant difference was found among the groups regarding the expression of genes related to osteogenesis (Alpl, Runx2),
It has been previously shown that Low-Magnitude High-Frequency Vibration (LMHFV) is able to enhance ovariectomy-induced osteoporotic fracture healing in rats. Fracture healing begins with the inflammatory stage, and all subsequent stages are regulated by the infiltration of immune cells such as macrophages and the release of inflammatory cytokines including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-10 (IL-10). Therefore, the aim of this study was to investigate the effect of LMFHV treatment on the inflammatory response in osteoporotic fracture healing. In this study, ovariectomy-induced osteoporotic and sham-operated closed-femoral fracture SD-rats were randomized into three groups: sham control (SHAM), ovariectomized control (OVX-C) or ovariectomized vibration (OVX-V) (n=36, n=6 per group per time point). LMHFV (35Hz, 0.3g) was given 20 min/day and 5 days/week to OVX-V group. SHAM operation and ovariectomy were performed at 6-month and closed femoral fracture was performed at 9-month. Callus morphometry was determined by callus width from weekly radiography. Local expressions of inducible nitric oxide synthase (iNOS) (macrophage M1 marker), CD206 (macrophage M2 marker), TNF-α, IL-6 and IL-10 were detected by immunohistochemistry and quantified by colour threshold in ImageJ, assessed at weeks 1 and 2 post-fracture. Significant difference between groups was considered at p≤0.05 by one-way ANOVA. Callus formation was higher in OVX-V than that of OVX-C as shown by callus width at weeks 1 and 2 (p=0.054 and 0.028, respectively). Immunohistochemistry results showed that CD206 positive signal and the M2/M1 ratio which indicates the progression of macrophage polarization were significantly higher in OVX-V rats (p=0.053 and 0.049, respectively) when compared to OVX-C at week 1. Area fraction of TNF-α positive signal was significantly higher in SHAM and OVX-V rats at week 1 (p=0.01 and 0.033, respectively). IL-6 signal was also significantly higher in SHAM and OVX-V groups at week 1 (p=0.004 and 0.029, respectively). IL-10 expression was significantly lower in SHAM and OVX-V groups at week 1 (p=0.013 and 0.05, respectively). Here we have shown that LMHFV treatment promoted the shift from pro-inflammatory stage towards anti-inflammatory stage earlier. It has been reported that the polarization of pro-inflammatory macrophages M1 to anti-inflammatory macrophages M2 was indicative of the endochondral ossification process in the long bone fracture model. Besides, we found that LMHFV treatment enhanced pro-inflammatory markers of TNF-α and IL-6 and suppressed anti-inflammatory marker of IL-10 at week 1, showing that inflammatory response was enhanced at week 1 post-fracture. These inflammatory cytokines involved in fracture healing were shown to coordinate different fracture healing processes such as mesenchymal stem cell recruitment and
Purpose. Angiogenesis and osteogenesis are essential for bone growth, fracture repair, and bone remodeling. VEGF has an important role in bone repair by promoting
Background. Despite promising results have shown by osteogenic cell-based demineralized bone matrix composites, they need to be optimized for grafts that act as structural frameworks in load-bearing defects. The purpose of this experiment is to determine the effect of bone marrow mesenchymal stem cells seeding on partially demineralized laser-perforated structural allografts that have been implanted in critical femoral defects. Materials and Methods. Thirty-two wistar rats were divided into four groups according to the type of structural bone allograft; the first: partially demineralized only (Donly), the second: partially demineralized stem cell seeded (DST), the third: partially demineralized laser-perforated (DLP), and the fourth: partially demineralized laser-perforated and stem cell seeded (DLPST). Trans-cortical holes were achieved in four rows of three holes approximated cylindrical holes 0.5 mm in diameter, with centres 2.5 mm apart. P3 MSCs were used for graft seeding. Histologic and histomorphometric analysis were performed at 12 weeks. Results. DLP grafts had the highest woven bone formation, where most parts of laser pores were completely healed by woven bone. DST and DLPST grafts surfaces had extra vessel-ingrowth-like porosities. Furthermore, in the DLPST grafts, a distinct bone formation at the interfaces was noted. Conclusion. This study indicated that surface changes induced by laser perforation, accelerated
Osteoarthritis (OA) is the most common form of arthritis worldwide. It is a major cause of disability in the adult population with its prevalence expected to increase dramatically over the next 20 years. Although current therapies can alleviate symptoms and improve function in early course of the disease, OA inevitably progresses to end-stage disease requiring total joint arthroplasty. Mesenchymal stromal cells (MSCs) have emerged as a candidate cell type with great potential for intra-articular (IA) repair therapy. However, there is still a considerable lack of knowledge concerning their behaviour, biology and therapeutic effects. To start addressing this, we explored the secretory profile of bone marrow derived MSCs in early and end-stage knee OA synovial fluid (SF). Subjects were recruited and categorised into early [Kellgren-Lawrence (KL) grade I and II, n=12] and end-stage (KL grade III and IV, n=11) knee OA groups. The SF proteome of early and end-stage OA was tested before and three days after the addition of bone marrow MSCs (16.5×10^3, single donor) using multiplex ELISA (64 cytokines) and mass spectrometry (302 proteins detected). Non parametric Wilcoxon-signed rank test for paired samples was used to compare the levels of proteins before and after addition of MSCs in early and end-stage knee OA SF. Significant differences were determined after multiple comparisons correction (FDR) with a p<0.05. Gender distribution and BMI were not statistically different between the two cohorts (p>0.05). However, patients in early knee OA cohort were significantly younger (44.7 years, SD=7.1) than patients in the end-stage cohort (58.6 years, SD=4.4; p<0.05). In both early and end-stage knee OA, MSCs increased the levels of VEGF-A (by 320.24 pg/mL), IL-6 (by 826.78 pg/mL) and IL-8 (by 128.85 pg/mL), factors involved in
Background. Osteoarthritis (OA) has been described as a non-inflammatory arthritis and yet the choice of drug treatment is NSAIDs. Aim. To test the hypothesis that cytokines and chemokines are associated with inflammation in OA. Methods. Synovium biopsy and synovial fluid of 17 patients undergoing total knee arthroplasty (TKR) were sampled at the onset of their surgery. Histology of synovium and immunoassay of synovial fluid were conducted. A 3 point scale, 3 being the most cellular, was used to assess the cellularity of synovium histology slides, a parameter known to correlate with several markers of OA. Synovial fluid was analysed using a multi-anylate fluorescent immunoassay. In brief, cytokines and chemokines associated with inflammation were quantified, namely IL-12, TNF, IL-10, IL-6, IL-1, IL-8. Results. The 3 point scale used to describe the cellularity of the synovium placed the majority in groups 2 and 3. Low levels (<120 pg/ml) of IL-12, IL-10, IL-1 and TNF were measured in all 3 cellularity groups. Markedly elevated values of IL-6 and IL-8 were measured in the synovial fluid of knees with the most cellular synovium (maximum values were 8325 pg/ml and 1540 pg/ml respectively). Conclusion. Elevated levels of IL-6 are associated with bone resorption, being clinically linked with aseptic loosening. IL-8 is capable of promoting
Ten percent of fractures end in delayed or non-union. NSAIDs have been linked to an inhibitory action on fracture repair for three decades yet the mechanism of action remains to be elucidated. Cancer research has identified that NSAIDs impede cell proliferation by inhibiting
Background. Synthetic interbody spinal fusion devices are used to restore and maintain disc height and ensure proper vertebral alignment. These devices are often filled with autograft bone to facilitate bone bridging through the device while providing mechanical stability. Nonporous polyetheretherketone (PEEK) devices are widely used clinically for such procedures. 1. Trabecular Metal devices are an alternative, fabricated from porous tantalum. It was hypothesized that the porous Trabecular Metal device would better maintain autograft viability through the center of the device, the ‘graft hole’ (GH). Methods. Twenty-five goats underwent anterior cervical discectomy and fusion using a Trabecular Metal or PEEK device for 6, 12 or 26 weeks. The GH of each device was filled with autograft bone morsels harvested from the animal at implantation. Fluorochrome labeling oxytetracycline was administered to the animals and used to determine bone viability in the device regions. Following necropsy, the vertebral segments were embedded in poly(methyl methacrylate) sectioned and analyzed using fluorescence and backscatter electron (BSE) imaging. The percent of bone tissue present within the GH was measured as a volume percent using BSE images (Fig. 1). Results. Bone percent analysis demonstrated that there was no significant difference (p<0.05) in volume of bone tissue within the GH of the two devices at 6 and 26 weeks (Fig. 2). At 12 weeks the animals implanted with the Trabecular Metal device had significantly greater volumes of bone within the GH region. Viable bone was observed in the host bone region and periprosthetic to the implant of all PEEK (n=12) and Trabecular Metal (n=12) animals within the study, determined by the presence of fluorescent labels (Fig. 3). Viable bone was also observed in the GH region of all animals with a Trabecular Metal device. However, only 5 of 12 PEEK animals showed bone viability within the GH (2 at 12 weeks and 3 at 26 weeks). A Fisher's exact comparison of the number of animals with viable bone in the GH showed a significant difference between the two devices, p<0.05. Conclusion. Autograft viability was better maintained within the GH for the porous Trabecular Metal device compared to the PEEK device. Although the amount of bone tissue within the GH of the PEEK devices was determined to have no significant difference compared to the Trabecular Metal devices at 6 and 26 weeks, the GH bone tissue was not viable in a number of the PEEK animals at each time point. The interconnected network and high volume porosity of the Trabecular Metal device may have allowed for fluid exchange,
The ability of mesenchymal stem cells (MSCs)
to differentiate Despite their increasing application in clinical trials, the
origin and role of MSCs in the development, repair and regeneration
of organs have remained unclear. Until recently, MSCs could only
be isolated in a process that requires culture in a laboratory;
these cells were being used for tissue engineering without understanding
their native location and function. MSCs isolated in this indirect
way have been used in clinical trials and remain the reference standard
cellular substrate for musculoskeletal engineering. The therapeutic
use of autologous MSCs is currently limited by the need for In this annotation we provide an update on the recent developments
in the understanding of the identity of MSCs within tissues and
outline how this may affect their use in orthopaedic surgery in
the future. Cite this article: