The multifunctional adhesion molecule CD44 is a major cell-surface receptor for hyaluronic acid (HUA). Recent data suggest that it may also bind the ubiquitous bone-matrix protein, osteopontin (OPN). Because OPN has been shown to be a potentially important protein in bone remodelling, we investigated the hypothesis that OPN interactions with the CD44 receptor on bone cells participate in the regulation of the healing of fractures. We examined the spatial and temporal patterns of expression of OPN and CD44 in healing fractures of rat femora by in situ hybridisation and immunohistochemistry. We also localised HUA in the
The Dkk3-derived cells represent a branch of the periosteal mesenchymal lineage that produces fibrocartilage as well as regenerating the periosteal structures. Mesenchymal progenitor cells are capable of generating a wide variety of mature cells that constitute the connective tissue system. Our Laboratory has been developing SMAA GFP reporter mice to prove to be an effective tool for identifying these cells prior to the expression of markers of differentiation characteristic of bone, fat, muscular blood vessels or fibrocartilage. Dkk3 was chosen as a candidate reporter because microarray of SMAA-sorted cells culture indicated high expression of this non-canonical anti-Wnt factor, which was not anticipated in a culture with strong osteogenic potential.Summary Statement
Introduction
Virtual mechanical testing is a method for measuring bone healing using finite element models built from computed tomography (CT) scans. Previously, we validated a dual-zone material model for ovine
Integrin α2β1 is one of the major transmembrane receptors for fibrillary collagen. In native bone we could show that the absence of this protein led to a protective effect against age-related osteoporosis. The objective of this study was to elucidate the effects of integrin α2β1 deficiency on fracture repair and its underlying mechanisms. Standardised femoral fractures were stabilised by an intramedullary nail in 12 week old female C57Bl/6J mice (wild type and integrin α2. -/-. ). After 7, 14 and 28 days mice were sacrificed. Dissected femura were subjected to µCT and histological analyses. To evaluate the biomechanical properties, 28-day-healed femura were tested in a torsional testing device. Masson goldner staining, Alizarin blue, IHC and IF staining were performed on paraffin slices. Blood serum of the animals were measured by ELISA for BMP-2. Primary osteoblasts were analysed by in/on-cell western technology and qRT-PCR. Integrin α2β1 deficient animals showed earlier transition from cartilaginous callus to mineralized
Mice are increasingly used for fracture healing research because of the possibility to use transgenic animals to conduct research on the molecular level. Mice from both sexes can be used, however, there is no consensus in the literature if fracture healing differs between female and male mice. Therefore, the aim of the present study was to analyze the similarities and differences in endochondral fracture healing between female and male C57BL/6J mice, since this mouse strain is mainly used in bone research. For that purpose, 12-weeks-old female and male mice received a standardized femur midshaft osteotomy stabilized by an external fixator. Mice were euthanized 10 and 21 days after fracture and bone regeneration was analyzed by biomechanical testing, µCT analysis, histology, immunohistochemistry and gene expression analysis. At day 21, male mice displayed a significantly larger
Objectives. Ubiquitin E3 ligase-mediated protein degradation regulates osteoblast function. Itch, an E3 ligase, affects numerous cell functions by regulating ubiquitination and proteasomal degradation of related proteins. However, the Itch-related cellular and molecular mechanisms by which osteoblast differentiation and function are elevated during bone fracture repair are as yet unknown. Methods. We examined the expression levels of E3 ligases and NF-κB members in callus samples during bone fracture repair by quantitative polymerase chain reaction (qPCR) and the total amount of ubiquitinated proteins by Western blot analysis in wild-type (WT) mice. The expression levels of osteoblast-associated genes in
Secondary bone healing is impacted by the extent of interfragmentary motion at the fracture site. It provides mechanical stimulus that is required for the formation of
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
Summary Statement. This study demonstrated that Sclerostin monoclonal antibody (Scl-Ab) enhanced bone healing in the rat osteotomy model. Scl-Ab increased callus size, callus bone volume fraction, rate of callus bone formation and
Fracture healing is an issue that has not yet been fully elucidated. It is generally accepted in the literature that head trauma accelerates fracture healing and causes higher volume callus tissue. Recent studies have examined the relationship between head trauma and fracture healing more molecularly. Based on this research; the aim of this study is to show the effect of head trauma on fracture healing radiologically and histologically and to investigate the relationship between serum β-Catenin level and fracture healing with the experiment we performed on rats. A total of 36 Wistar Albino female rats with a mean age of 24 weeks were included in the study with the permission of Mersin University Animal Experiments Local Ethics Committee. Six rats in the first group were not traumatized and their blood samples were collected on the day of the experiment started, end of the third week and end of the sixth week. In the second group, only head trauma was performed and blood samples were collected at the end of the third and sixth weeks. In the third group, only open femoral fracture model was applied, blood samples were collected at the third and sixth weeks and AP and Lateral radiographs of the fractured femurs were taken. After sacrification, femurs were dissected from the surrounding soft tissues and subjected to histological examination. In the fourth group, both head trauma and open femur fracture model were applied, blood samples were collected at the end of third and sixth weeks and AP and Lateral radiographs of the fractured femurs were taken. After sacrification, femurs were dissected from the surrounding soft tissues and subjected to histological examination. The expression level of β-Catenin was measured by PCR from all blood samples. Direct radiographs of the third and fourth groups at 3 and 6 weeks were evaluated by two orthopedists according to Rust and Lane & Sandhu scoring system. The histomorphometric examination was performed by evaluating the Huo scoring and the ratio of
Myostatin (GDF-8) is known to play an important role in muscle regeneration, and myostatin is also expressed during the early phases of fracture healing. In this study we used fluorescent immunohistochemistry to define the temporal and spatial localization of myostatin during muscle and bone repair following deep penetrant injury in a mouse model. We then used hydrogel delivery of exogenous myostatin in the same injury model to determine the effects of myostatin exposure on muscle and bone healing. Results show that while myostatin was constitutively expressed in the cytoplasm of intact skeletal muscle fibers, a pool of intense myostatin staining was observed amongst injured skeletal muscle fibers 12-24 hours post-surgery. Myostatin was also expressed in the soft callus chondrocytes 4 days following osteotomy. Hydrogel delivery of 10 or 100 ug/ml recombinant myostatin decreased
We studied the effect of vitamin C on fracture healing in the elderly. A total of 80 elderly Osteogenic Disorder Shionogi rats were divided into four groups with different rates of vitamin C intake. A closed bilateral fracture was made in the middle third of the femur of each rat. Five weeks after fracture the femora were analysed by mechanical and histological testing. The groups with the lower vitamin C intake demonstrated a lower mechanical resistance of the healing callus and a lower histological grade. The vitamin C levels in blood during healing correlated with the torque resistance of the callus formed (r = 0.525). Therefore, the supplementary vitamin C improved the mechanical resistance of the
Objectives. Recent studies have shown that modulating inflammation-related
lipid signalling after a bone fracture can accelerate healing in
animal models. Specifically, decreasing 5-lipoxygenase (5-LO) activity
during fracture healing increases cyclooxygenase-2 (COX-2) expression
in the
Introduction. Subchondral insufficiency fracture (SIF) needs to be differentiated from osteonecrosis. The purpose of this study was to evaluate the imaging and histopathological findings of patients 60 years or older with radiological evidence of subchondral collapse of the femoral head. Methods. We reviewed 77 consecutive hips in 56 patients aged 60 years or older, which showed subchondral collapse on radiographs. According to the shapes of low-intensity bands on T1-weighted images of magnetic resonance imaging (MRI), the patients were divided into 2 groups. Group A showed a concavity of the articular surface: characteristic appearances of osteonecrosis, and Group B showed an irregular convexity of the articular surface: characteristic appearances of SIF. Results. Thirty-four patients (60.7%) were categorized as Group A and 22 patients (39.3%) as Group B. All the cases in Group A had a history of corticosteroid intake or alcohol abuse. In Group B, the proportion of women as well as unilateral involvement was significantly higher than those in Group A. In 30 cases with histopathological examination, 16 were in Group A and 14 were in Group B. All the cases in Group A demonstrated characteristic features of osteonecrosis. In Group B, the band on T1-weighted images corresponded to viable reparative tissue including granulation tissue and
Introduction. Amino acids like arginine and lysine have been suggested to hasten the process of fracture healing by improving the local blood supply, supplementing growth factors, and improving collagen synthesis. We studied the role of lysine and arginine in the fracture repair process with regard to the rate of healing, probable mechanisms involved in the process, and mutual synergism between these agents. Materials and methods. In an experimental study, 40 rabbits were subjected to ulnar osteotomy. They were distributed in control (14) and test groups (26). Twenty-six animals in the test group were fed with a diet rich in lysine and arginine. Both the groups were followed radiologically and histologically till union. Results. Ten weeks postoperatively, there was difference evident radiologically between those supplemented with lysine and arginine, indicating that these components enhance the healing in the later part of bone remodeling, canal restoration, and medullary as well as cortical continuity and repair. X-rays obtained at weeks 9, 10, and 12 in both the groups showed statistical significance. These findings showed that healing is better in the test group in terms of increased vascularity in the early part of healing, i.e., at approx. 2–3 weeks and in terms of bone matrix, Haversian system formation, and cortical repair in the later part of healing, i.e., at approx. 9–12 weeks between the two groups. There was better healing of osteotomy in terms of better vascularization, callus formation, and mineralization in the test group. The time of healing in the test group was reduced by a period of 2 weeks. Discussion. NO is expressed during fracture healing in rats and humans, as after fracture, mRNA, protein, and enzymatic activity iNOS have been identified at the
Summary Statement. Parathytorid hormone-related protein (107–111) loaded onto biopolymer-coated nanocrystalline hydroxyapatite (HA. Glu. ) improves the bone repair in a cavitary defect in rat tibiae. Introduction. Biopolymer-coated nanocrystalline hydroxyapatite (HA. Glu. ) made as macroporous foams are promising candidates as scaffolds for bone tissue engineering applications. They exhibit optimal features, promoting internalization, proliferation and differentiation of osteoprogenitors, with an adequate cell colonization over the entire scaffold surface. Parathyroid hormone-related protein (PTHrP) is an important modulator of bone formation. Its 107–111 epitope (osteostatin) exhibits osteogenic properties at least in part by directly acting on osteoblasts. The main aim of this study was to evaluate whether osteostatin loading into HA. Glu. scaffolds might improve their bone regeneration capacity. Materials and Methods. HA. Glu. scaffolds were prepared as previously described (Sánchez-Salcedo S et al. J. Mater. Chem. 2010; 20:6956-61). Osteostatin was adsorbed onto HA. Glu. material by dipping into a solution containing this peptide at 100 nM (in phosphate-buffered saline, pH 7.4), following a standard protocol. We performed a cavitary defect (2 mm in diameter and 3 mm in depth) in both distal tibial metaphysis using a drill under general anesthesia in male Wistar rats (n=8) of 6 months of age. Unloaded HA. Glu. material (7 mg) was implanted into left tibial defects, whereas rigth tibial defects received the osteostatin-loaded material. Animals were sacrificed after 4 weeks for histological, μ-computerised tomography and gene expression analysis of the callus. Our protocol was approved by the Institutional Animal Care and Use Committee at the IIS-FJD. Mouse osteoblastic MC3T3-E1 cells were grown in differentiation medium (α-MEM with 10% fetal bovine serum, 50 µg/ml ascorbic acid, and 10 mM β-glycerolphosphate), in the presence or absence of HA. Glu. material with or without osteostatin. Cell viability (assessed by trypan blue staining), alkaline phosphatase (ALP) activity and mineralization (alizarin red) were analyzed at different culture times. Results. The mean uptake of osteostatin by HA. Glu. scaffolds was about 60 % (representing 0.7 μg/implanted scaffold) after 24 h of loading, and they released a mean of 80 % of loaded peptide to the surrounding medium within 1–24 h. At 4 weeks, this osteostatin-containing HA. Glu. material significantly increased the bone volumen fraction and trabecular thickness of regenerating bone in the tibial methaphysis, compared to those observed with unloaded HAGlu scaffolds. In addition, osteostatin-coated HA. Glu. scaffolds increased (2-fold) the gene expression of osteocalcin and vascular cell adhesion molecule 1, but decreased (2-fold) that of the Wnt inhibitors, SOST and Dickkopf homolog 1 (DKK-1) in the
We investigated the effects on fracture healing of two up-regulators of inducible nitric oxide synthase (iNOS) in a rat model of an open femoral osteotomy: tadalafil, a phosphodiesterase inhibitor, and the recently reported nutraceutical, COMB-4 (consisting of L-citrulline, Paullinia cupana, ginger and muira puama), given orally for either 14 or 42 days. Unilateral femoral osteotomies were created in 58 male rats and fixed with an intramedullary compression nail. Rats were treated daily either with vehicle, tadalafil or COMB-4. Biomechanical testing of the healed fracture was performed on day 42. The volume, mineral content and bone density of the callus were measured by quantitative CT on days 14 and 42. Expression of iNOS was measured by immunohistochemistry.Objectives
Materials and Methods
This study was designed to test the hypothesis
that the sensory innervation of bone might play an important role
in sensing and responding to low-intensity pulsed ultrasound and
explain its effect in promoting fracture healing. In 112 rats a
standardised mid-shaft tibial fracture was created, supported with
an intramedullary needle and divided into four groups of 28. These
either had a sciatic neurectomy or a patellar tendon resection as
control, and received the ultrasound or not as a sham treatment.
Fracture union, callus mineralisation and remodelling were assessed using
plain radiography, peripheral quantitative computed tomography and
histomorphology. Daily ultrasound treatment significantly increased the rate of
union and the volumetric bone mineral density in the fracture callus
in the neurally intact rats (p = 0.025), but this stimulating effect
was absent in the rats with sciatic neurectomy. Histomorphology
demonstrated faster maturation of the callus in the group treated
with ultrasound when compared with the control group. The results
supported the hypothesis that intact innervation plays an important
role in allowing low-intensity pulsed ultrasound to promote fracture
healing.
We have previously shown that joint distraction and movement with a hinged external fixation device for 12 weeks was useful for repairing a large articular cartilage defect in a rabbit model. We have now investigated the results after six months and one year. The device was applied to 16 rabbits who underwent resection of the articular cartilage and subchondral bone from the entire tibial plateau. In group A (nine rabbits) the device was applied for six months. In group B (seven rabbits) it was in place for six months, after which it was removed and the animals were allowed to move freely for an additional six months. The cartilage remained sound in all rabbits. The areas of type II collagen-positive staining and repaired soft tissue were larger in group B than in group A. These findings provide evidence of long-term persistence of repaired cartilage with this technique and that weight-bearing has a positive effect on the quality of the cartilage.
Conventional non-steroidal anti-inflammatory drugs (NSAIDs) and newer specific cyclo-oxygenase-2 (cox-2) inhibitors are commonly used in musculoskeletal trauma and orthopaedic surgery to reduce the inflammatory response and pain. These drugs have been reported to impair bone metabolism. In reconstruction of the anterior cruciate ligament the hamstring tendons are mainly used as the graft of choice, and a prerequisite for good results is healing of the tendons in the bone tunnel. Many of these patients are routinely given NSAIDs or cox-2 inhibitors, although no studies have elucidated the effects of these drugs on tendon healing in the bone tunnel. In our study 60 female Wistar rats were randomly allocated into three groups of 20. One received parecoxib, one indometacin and one acted as a control. In all the rats the tendo-Achillis was released proximally from the calf muscles. It was then pulled through a drill hole in the distal tibia and sutured anteriorly. The rats were given parecoxib, indometacin or saline intraperitoneally twice daily for seven days. After 14 days the tendon/bone-tunnel interface was subjected to mechanical testing. Significantly lower maximum pull-out strength (p <
0.001), energy absorption (p <
0.001) and stiffness (p = 0.035) were found in rats given parecoxib and indometacin compared with the control group, most pronounced with parecoxib.