Hematopoietic Wnts Modulate Endochondral Ossification During Fracture Healing.

Wnt signaling plays a critical role in bone formation, homeostasis, and injury repair. Multiple cell types in bone have been proposed to produce the Wnts required for these processes. The specific role of Wnts produced from cells of hematopoietic origin has not been previously characterized. Here, we examined if hematopoietic Wnts play a role in physiological musculoskeletal development and in fracture healing. Wnt secretion from hematopoietic cells was blocked by genetic knockout of the essential Wnt modifying enzyme PORCN, achieved by crossing Vav-Cre transgenic mice with Porcnflox mice. Knockout mice were compared with their wild-type littermates for musculoskeletal development including bone quantity and quality at maturation. Fracture healing including callus quality and quantity was assessed in a diaphyseal fracture model using quantitative micro computer-assisted tomographic scans, histological analysis, as well as biomechanical torsional and 4-point bending stress tests. The hematopoietic Porcn knockout mice had normal musculoskeletal development, with normal bone quantity and quality on micro-CT scans of the vertebrae. They also had normal gross skeletal dimensions and normal bone strength. Hematopoietic Wnt depletion in the healing fracture resulted in fewer osteoclasts in the fracture callus, with a resultant delay in callus remodeling. All calluses eventually progressed to full maturation. Hematopoietic Wnts, while not essential, modulate osteoclast numbers during fracture healing. These osteoclasts participate in callus maturation and remodeling. This demonstrates the importance of diverse Wnt sources in bone repair.

Expression of Musashi-1 Increases in Bone Healing.

Musashi-1 (MSI1) is an RNA-binding protein that regulates progenitor cells in adult and developing organisms to maintain self-renewal capacities. The role of musashi-1 in the bone healing environment and its relation with other osteogenic factors is unknown. In the current study, we analyze the expression of MSI1 in an experimental model of rat femoral bone fractures. We also analyze the relation between MSI1 expression and the expression of two osteogenic markers: periostin (POSTN) and runt-related transcription factor 2 (RUNX2). We use histological, immunohistochemical, and qPCR techniques to evaluate bone healing and the expression of MSI1, POSTN, and RUNX2 over time (4, 7, and 14 days). We compare our findings with non-fractured controls. We find that in bone calluses, the number of cells expressing MSI1 and RUNX2 increase over time and the intensity of POSTN expression decreases over time. Within bone calluses, we find the presence of MSI1 expression in mesenchymal stromal cells, osteoblasts, and osteocytes but not in hypertrophic chondrocytes. After 14 days, the expression of MSI1, POSTN, and RUNX2 was significantly correlated. Thus, we conclude that musashi-1 potentially serves in the osteogenic differentiation of mesenchymal stromal cells and bone healing. Therefore, further studies are needed to determine the possibility of musashi-1's role as a clinical biomarker of bone healing and therapeutic agent for bone regeneration.

Extracellular matrix and wall composition are diverse in the organogenic and non-organogenic calli of Actinidia arguta.

KEY MESSAGE: Differences in the composition and the structural organisation of the extracellular matrix correlate with the morphogenic competence of the callus tissue that originated from the isolated endosperm of kiwifruit. The chemical composition and structural organisation of the extracellular matrix, including the cell wall and the layer on its surface, may correspond with the morphogenic competence of a tissue. In the presented study, this relationship was found in the callus tissue that had been differentiated from the isolated endosperm of the kiwiberry, Actinidia arguta. The experimental system was based on callus samples of exactly the same age that had originated from an isolated endosperm but were cultured under controlled conditions promoting either an organogenic or a non-organogenic pathway. The analyses which were performed using bright field, fluorescence and scanning electron microscopy techniques showed significant differences between the two types of calli. The organogenic tissue was compact and the outer walls of the peripheral cells were covered with granular structures. The non-organogenic tissue was composed of loosely attached cells, which were connected via a net-like structure. The extracellular matrices from both the non- and organogenic tissues were abundant in pectic homogalacturonan and extensins (LM19, LM20, JIM11, JIM12 and JIM20 epitopes), but the epitopes that are characteristic for rhamnogalacturonan I (LM5 and LM6), hemicellulose (LM25) and the arabinogalactan protein (LM2) were detected only in the non-organogenic callus. Moreover, we report the epitopes, which presence is characteristic for the Actinidia endosperm (LM21 and LM25, heteromannan and xyloglucan) and for the endosperm-derived cells that undergo dedifferentiation (loss of LM21 and LM25; appearance or increase in the content of LM5, LM6, LM19, JIM11, JIM12, JIM20, JIM8 and JIM16 epitopes).

Deletion of Runx1 in osteoclasts impairs murine fracture healing through progressive woven bone loss and delayed cartilage remodeling.

Conditional deletion of the transcription factor Runt-related transcription factor 1 (Runx1) in myeloid osteoclast precursors promotes osteoclastogenesis and subsequent bone loss. This study posits whether Runx1 regulates clastic cell-mediated bone and cartilage resorption in the fracture callus. We first generated mice, in which Runx1 was conditionally abrogated in osteoclast precursors (LysM-Cre;Runx1F/F ; Runx1 cKO). Runx1 cKO and control mice were then subjected to experimental mid-diaphyseal femoral fractures. Our study found differential resorption of bony and calcified cartilage callus matrix by osteoclasts and chondroclasts within Runx1 cKO calluses, with increased early bony callus resorption and delayed calcified cartilage resorption. There was an increased number of osteoclasts and chondroclasts in the chondro-osseous junction of Runx1 cKO calluses starting at day 11 post-fracture, with minimal woven bone occupying the callus at day 18 post-fracture. LysM-Cre;Runx1F/F mutant mice had increased bone compliance at day 28, but their strength and work to failure were comparable with controls. Taken together, these results indicate that Runx1 is a critical transcription factor in controlling osteoclastogenesis that negatively regulates bone and cartilage resorption in the fracture callus. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:1007-1015, 2020.

Decellularized Cartilage Directs Chondrogenic Differentiation: Creation of a Fracture Callus Mimetic.

Complications that arise from impaired fracture healing have considerable socioeconomic implications. Current research in the field of bone tissue engineering predominantly aims to mimic the mature bone tissue microenvironment. This approach, however, may produce implants that are intrinsically unresponsive to the cues present during the initiation of fracture repair. As such, this study describes the development of decellularized xenogeneic hyaline cartilage matrix in an attempt to mimic the initial reparative phase of fracture repair. Three approaches based on vacuum-assisted osmotic shock (Vac-OS), Triton X-100 (Vac-STx), and sodium dodecyl sulfate (Vac-SDS) were investigated. The Vac-OS methodology reduced DNA content below 50 ng/mg of tissue, while retaining 85% of the sulfate glycosaminoglycan content, and as such was selected as the optimal methodology for decellularization. The resultant Vac-OS scaffolds (decellularized extracellular matrix [dcECM]) were also devoid of the immunogenic alpha-Gal epitope. Furthermore, minimal disruption to the structural integrity of the dcECM was demonstrated using differential scanning calorimetry and fluorescence lifetime imaging microscopy. The biological integrity of the dcECM was confirmed by its ability to drive the chondrogenic commitment and differentiation of human chondrocytes and periosteum-derived cells, respectively. Furthermore, histological examination of dcECM constructs implanted in immunocompetent mice revealed a predominantly M2 macrophage-driven regenerative response both at 2 and 8 weeks postimplantation. These findings contrasted with the implanted native costal cartilage that elicited a predominantly M1 macrophage-mediated inflammatory response. This study highlights the capacity of dcECM from the Vac-OS methodology to direct the key biological processes of endochondral ossification, thus potentially recapitulating the callus phase of fracture repair.

Osteoclast depletion with clodronate liposomes delays fracture healing in mice.

Osteoclasts are abundant within the fracture callus and also localize at the chondro-osseous junction. However, osteoclast functions during fracture healing are not well defined. Inhibition of osteoclast formation or resorptive activity impairs callus remodeling but does not prevent callus formation. Interestingly, though anti-osteoclast therapies differentially affect resolution of callus cartilage into bone. Treatments that inhibit osteoclast formation or viability tend to impair callus cartilage resolution, while treatments that target inhibition of bone resorption generally do not affect callus cartilage resolution. Here, we tested whether depletion of osteoclasts by systemic treatment with clodronate liposomes would similarly impair callus cartilage resolution. ICR mice were treated by intraperitoneal injections of clodronate-laden liposomes or control liposomes and subjected to closed femur fracture. Femurs were resected at multiple times after fracture and analyzed by radiography, histology, and mechanical testing to determine effects on healing. Clodronate liposome treatment did not prevent callus formation. However, radiographic scoring indicated that clodronate liposome treatment impaired healing. Clodronate liposome treatment significantly reduced callus osteoclast populations and delayed resolution of callus cartilage. Consistent with continued presence of callus cartilage, torsional mechanical testing found significant decreases in callus material properties after 28 days of healing. The results support a role for osteoclasts in the resolution of callus cartilage into bone. Whether the cartilage resolution role for osteoclasts is limited to simply resorbing cartilage at the chondro-osseous junction or in promoting bone formation at the chondro-osseous junction through another mechanism, perhaps similar to the reversal process in bone remodeling, will require further experimentation. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1699-1706, 2017.

The osteogenic potential of human bone callus.

Bone callus, generated during fracture healing, is commonly discarded during surgical procedures. The aim of this study was to investigate the osteogenic potential of bone callus and its possible use as autograft material for patients needing bone grafts. Histology, immunohistochemistry, micro-computed tomography, and biomechanics were performed to examine osteogenic cells, osteoinductive factors, and the osteoconductive structure of bone callus. Alkaline phosphatase-positive osteoblasts, osteoinductive factors (including BMP2, FGF2, TGFB1, and IGF1), and a porous structure were found in bone callus. Early-stage callus (within 3 months after fracture) presented significantly improved osteogenic properties compared to medium- (3-9 months) and late-stage (longer than 9 months) callus. The results revealed that bone callus induced new bone formation in a nude mouse model. Early-stage callus showed better performance to medium- and late-stage callus in the induction of new bone formation at both 8 and 12 weeks. These findings indicated that bone callus, especially early-stage callus, possesses osteogenic potential and can potentially serve as an alternative source of material for bone grafts.

The effect of femoral nerve block on fracture healing via expressions of growth factors and ß-catenin.

INTRODUCTION: Many patients of all ages are admitted to hospital due to bone fractures. The etiology of fracture has a very wide spectrum, ranging from motor accidents to pathological conditions such as tumors, osteoporosis, and others. Bone fracture healing is a well-programmed and well-organized process, but is also long and intractable. The outcome of this process is therefore affected by many factors, such as the patient's age, ethnicity, nutritional status, and extent of the fracture. At present, regional analgesic techniques are frequently applied in order to avoid the complications of systemic opioid administration, central block applications. Femoral block is one of the regional analgesic techniques frequently applied by anesthesiologists when the lower extremities are involved. In this study, we evaluated the effect of femoral nerve block on the healing of an experimental non-stabilized femur fracture via expression of TGF-ß, VEGF, and ß-catenin and bone histomorphometry in rats. MATERIAL AND METHODS: In the control group, only the femoral fracture was performed and the bone was not fixated, similarly as in other groups. In the One-Day Block group, a one-time femoral nerve block was applied after the femoral fracture. In the Three-Day Block group, a daily femoral nerve block was performed for three days after the femoral fracture. On Days 4, 7, and 13, femurs were excised. The bone sections were stained with hematoxylin-eosin to evaluate bone tissue and Safranin O to assess callus tissue, cartilaginous tissue, and new bone areas. TGF-ß, VEGF, and ß-catenin were assessed by immunohistochemistry. RESULTS: Histomorphometric analysis revealed that femoral block application had a positive impact on bone healing. TGF-ß expression in the One-Day and Three-Day Block Groups was significantly higher than in the control group at all times, as was also the case with VEGF expression. On day 13, ß-catenin expression was significantly higher in the Three-Day Block group than the others. CONCLUSIONS: The results of the study suggests that the applications of a femoral nerve block for perioperative analgesia, for either one day or three days, resulted in better and more rapid bone healing.

The ameloblastin extracellular matrix molecule enhances bone fracture resistance and promotes rapid bone fracture healing.

The extracellular matrix (ECM) provides structural support, cell migration anchorage, cell differentiation cues, and fine-tuned cell proliferation signals during all stages of bone fracture healing, including cartilaginous callus formation, callus remodeling, and bony bridging of the fracture gap. In the present study we have defined the role of the extracellular matrix protein ameloblastin (AMBN) in fracture resistance and fracture healing of mouse long bones. To this end, long bones from WT and AMBN(Δ5-6) truncation model mice were subjected to biomechanical analysis, fracture healing assays, and stem cell colony formation comparisons. The effect of exogenous AMBN addition to fracture sites was also determined. Our data indicate that lack of a functional AMBN in the bone matrix resulted in 31% decreased femur bone mass and 40% reduced energy to failure. On a cellular level, AMBN function inhibition diminished the proliferative capacity of fracture repair callus cells, as evidenced by a 58% reduction in PCNA and a 40% reduction in Cyclin D1 gene expression, as well as PCNA immunohistochemistry. In terms of fracture healing, AMBN truncation was associated with an enhanced and prolonged chondrogenic phase, resulting in delayed mineralized tissue gene expression and delayed ossification of the fracture repair callus. Underscoring a role of AMBN in fracture healing, there was a 6.9-fold increase in AMBN expression at the fracture site one week after fracture, and distinct AMBN immunolabeling in the fracture gap. Finally, application of exogenous AMBN protein to bone fracture sites accelerated callus formation and bone fracture healing (33% increase in bone volume and 19% increase in bone mineral density), validating the findings of our AMBN loss of function studies. Together, these data demonstrate the functional importance of the AMBN extracellular matrix protein in bone fracture prevention and rapid fracture healing.

Identification and characterization of an injury-induced skeletal progenitor.

The postnatal skeleton undergoes growth, remodeling, and repair. We hypothesized that skeletal progenitor cells active during these disparate phases are genetically and phenotypically distinct. We identified a highly potent regenerative cell type that we term the fracture-induced bone, cartilage, stromal progenitor (f-BCSP) in the fracture callus of adult mice. The f-BCSP possesses significantly enhanced skeletogenic potential compared with BCSPs harvested from uninjured bone. It also recapitulates many gene expression patterns involved in perinatal skeletogenesis. Our results indicate that the skeletal progenitor population is functionally stratified, containing distinct subsets responsible for growth, regeneration, and repair. Furthermore, our findings suggest that injury-induced changes to the skeletal stem and progenitor microenvironments could activate these cells and enhance their regenerative potential.

Osteoblast-Specific Loss of IGF1R Signaling Results in Impaired Endochondral Bone Formation During Fracture Healing.

Insulin-like growth factors (IGFs) are important local regulators during fracture healing. Although IGF1 deficiency is known to increase the risk of delayed union or non-union fractures in the elderly population, the underlying mechanisms that contribute to this defect remains unclear. In this study, IGF1 signaling during fracture healing was investigated in an osteoblast-specific IGF1 receptor (IGF1R) conditional knockout (KO) mouse model. A closed tibial fracture was induced in IGF1R(flox/flox) /2.3-kb α1(1)-collagen-Cre (KO) and IGF1R(flox/flox) (control) mice aged 12 weeks. Fracture callus samples and nonfractured tibial diaphysis were collected and analyzed by µCT, histology, immunohistochemistry, histomorphometry, and gene expression analysis at 10, 15, 21, and 28 days after fracture. A smaller size callus, lower bone volume accompanied by a defect in mineralization, bone microarchitectural abnormalities, and a higher cartilage volume were observed in the callus of these KO mice. The levels of osteoblast differentiation markers (osteocalcin, alkaline phosphatase, collagen 1α1) were significantly reduced, but the early osteoblast transcription factor runx2, as well as chondrocyte differentiation markers (collagen 2α1 and collagen 10α1) were significantly increased in the KO callus. Moreover, increased numbers of osteoclasts and impaired angiogenesis were observed during the first 15 days of fracture repair, but decreased numbers of osteoclasts were found in the later stages of fracture repair in the KO mice. Although baseline nonfractured tibias of KO mice had decreased trabecular and cortical bone compared to control mice, subsequent studies with mice expressing the 2.3-kb α1(1)-collagen-Cre ERT2 construct and given tamoxifen at the time of fracture and so starting with comparable bone levels showed similar impairment in fracture repair at least initially. Our data indicate that not only is the IGF1R in osteoblasts involved in osteoblast differentiation during fracture repair, but it plays an important role in coordinating chondrocyte, osteoclast, and endothelial responses that all contribute to the endochondral bone formation required for normal fracture repair.

Absence of substance P and the sympathetic nervous system impact on bone structure and chondrocyte differentiation in an adult model of endochondral ossification.

OBJECTIVE: Sensory and sympathetic nerve fibers (SNF) innervate bone and epiphyseal growth plate. The role of neuronal signals for proper endochondral ossification during skeletal growth is mostly unknown. Here, we investigated the impact of the absence of sensory neurotransmitter substance P (SP) and the removal of SNF on callus differentiation, a model for endochondral ossification in adult animals, and on bone formation. METHODS: In order to generate callus, tibia fractures were set in the left hind leg of wild type (WT), tachykinin 1-deficient (Tac1-/-) mice (no SP) and animals without SNF. Locomotion was tested in healthy animals and touch sensibility was determined early after fracture. Callus tissue was prepared for immunofluorescence staining for SP, neurokinin1-receptor (NK1R), tyrosine-hydroxylase (TH) and adrenergic receptors α1, α2 and ß2. At the fracture site, osteoclasts were stained for TRAP, osteoblasts were stained for RUNX2, and histomorphometric analysis of callus tissue composition was performed. Primary murine bone marrow derived macrophages (BMM), osteoclasts, and osteoblasts were tested for differentiation, activity, proliferation and apoptosis in vitro. Femoral fractures were set in the left hind leg of all the three groups for mechanical testing and µCT-analysis. RESULTS: Callus cells stained positive for SP, NK1R, α1d- and α2b adrenoceptors and remained ß2-adrenoceptor and TH-negative. Absence of SP and SNF did not change the general locomotion but reduces touch sensitivity after fracture. In mice without SNF, we detected more mesenchymal callus tissue and less cartilaginous tissue 5 days after fracture. At day 13 past fracture, we observed a decrease of the area covered by hypertrophic chondrocytes in Tac1-/- mice and mice without SNF, a lower number of osteoblasts in Tac1-/- mice and an increase of osteoclasts in mineralized callus tissue in mice without SNF. Apoptosis rate and activity of osteoclasts and osteoblasts isolated from Tac1-/- and sympathectomized mice were partly altered in vitro. Mechanical testing of fractured- and contralateral legs 21 days after fracture, revealed an overall reduced mechanical bone quality in Tac1-/- mice and mice without SNF. µCT-analysis revealed clear structural alteration in contralateral and fractured legs proximal of the fracture site with respect to trabecular parameters, bone mass and connectivity density. Notably, structural parameters are altered in fractured legs when related to unfractured legs in WT but not in mice without SP and SNF. CONCLUSION: The absence of SP and SNF reduces pain sensitivity and mechanical stability of the bone in general. The micro-architecture of the bone is profoundly impaired in the absence of intact SNF with a less drastic effect in SP-deficient mice. Both sympathetic and sensory neurotransmitters are indispensable for proper callus differentiation. Importantly, the absence of SP reduces bone formation rate whereas the absence of SNF induces bone resorption rate. Notably, fracture chondrocytes produce SP and its receptor NK1 and are positive for α-adrenoceptors indicating an endogenous callus signaling loop. We propose that sensory and sympathetic neurotransmitters have crucial trophic effects which are essential for proper bone formation in addition to their classical neurological actions.

Determination of suitable microspore stage and callus induction from anthers of kenaf (Hibiscus cannabinus L.).

Kenaf (Hibiscus cannabinus L.) is one of the important species of Hibiscus cultivated for fiber. Availability of homozygous parent lines is prerequisite to the use of the heterosis effect reproducible in hybrid breeding. The production of haploid plants by anther culture followed by chromosome doubling can be achieved in short period compared with inbred lines by conventional method that requires self pollination of parent material. In this research, the effects of the microspore developmental stage, time of flower collection, various pretreatments, different combinations of hormones, and culture condition on anther culture of KB6 variety of Kenaf were studied. Young flower buds with immature anthers at the appropriate stage of microspore development were sterilized and the anthers were carefully dissected from the flower buds and subjected to various pretreatments and different combinations of hormones like NAA, 2,4-D, Kinetin, BAP, and TDZ to induce callus. The best microspore development stage of the flower buds was about 6-8 mm long collected 1-2 weeks after flower initiation. At that stage, the microspores were at the uninucleate stage which was suitable for culture. The best callus induction frequency was 90% in the optimized semisolid MS medium fortified with 3.0 mg/L BAP + 3.0 mg/L NAA.

Shoot organogenesis from root-derived callus of Rhinacanthus nasutus (L.) Kurz. and assessment of clonal fidelity of micropropagted plants using RAPD analysis.

An efficient regeneration system was established for an ethnomedicinal shrub Rhinacanthus nasutus from root-derived callus organogenesis. The root segments were cultured on MS medium supplemented with various concentrations of Kn (1.0-4.0 µM) alone or in combination with IBA (0.2-0.6 µM) or 2, 4-D (0.5-1.5 µM). The optimum frequency (94%) of callus induction was recorded on MS medium supplemented with 3.0 µM Kn and 0.4 µM IBA. For shoot regeneration from callus, MS medium supplemented with different concentrations (1.0-7.0 µM) of BA or TDZ alone or in combination with NAA (0.2-1.0 µm) was employed. The highest frequency of shoot regeneration (91%) and mean number of shoots (28.3) were observed on MS medium supplemented with 5.0 µM BA and 0.7 µM NAA. The shoots were excised and cultured on MS medium with 4.0 µM IBA produced 3.4 roots per shoot in 88% cultures. Of the 65 plants transferred to soil 54 survived (83%). The plants were transferred to field after successful hardening. RAPD analysis of the regenerated plants showed high similarity with the mother plant.

Comparative bioactive studies between wild plant and callus culture of Tephrosia tinctoria pers.

Tephrosia tinctoria, a perennial under shrub of Fabaceae family, is endemic to Western Ghats. In this study, friable whitish yellow callus was developed after 45 days using Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid (2.0 mg/l) + 6-benzylaminopurine (0.5 mg/l) in various explants of T. tinctoria. The ethyl acetate extracts of leaf (LE), stem (SE), and root (RE) were compared with leaf (LCE), stem (SCE), and root (RCE) derived callus, for antioxidant and antiproliferative activities. The SE possessed the highest phenolic and flavonoid content among all the extracts tested and showed a significant antioxidant assays. The study of anticancer activity on human hepatocellular carcinoma (HepG2) cell line revealed that the callus extracts especially RCE possessed significant inhibition of cell growth (IC50 20 µg/ml) at 72 h treatment period on analysis with MTT assay. The apoptotic cell death was observed through DNA fragmentation analysis in HepG2 cells treated with the T. tinctoria extracts. The gas chromatography-mass spectrometry finger printing profile showed that more than 60 % percentage of metabolites are similar in both SE and SCE. The higher percentage area of antioxidant compound (stigmast-4-en-3-one) was observed in SE (2.01 %) and higher percentage area of anticancer compound (phenol, 2,4-bis(1,1-dimethylethyl)) in SCE (0.91 %). In addition to that, callus extracts contain squalene, which is used for target deliver and also used as anticancer drug. Thus, the present study revealed that the T. tinctoria has potent antioxidant and antiproliferative activity and the callus culture can be used for the production of the bioactive compounds due to the endemic nature of this plant.

Comparative antioxidant study of stem and stem induced callus of Phyllanthus fraternus webster-an important antiviral and hepatoprotective plant.

Phyllanthus fraternus is widely used in the cure of various liver diseases and possess antiviral properties especially against hepatitis virus. In the present study, evaluation of the antioxidant activity of stem and calli induced from stem has been done by different assays. Extraction was done by standard method in water and ethanol. Total antioxidant capacity was measured by 1, 1-diphenyl-2-picrylhydrazyl free radical scavenging method. Lipid peroxidation was measured in terms of thiobarbituric acid-reactive substances (TBARS) by using egg yolk homogenates as lipid-rich media, and superoxide radical scavenging activity was measured using riboflavin-light-nitro blue tetrazolium assay. Reducing power was determined on the basis of Fe(3+)-Fe(2+) transformation in the presence of the extract. In addition to the antioxidant activity, polyphenolic compounds like total phenolics and flavonoids were also measured by spectroscopic method. Results showed that the ethanolic extract of stem is more potent in antioxidant activity than its aqueous extract and ethanolic extract of calli. A significant correlation between antioxidant capacity and polyphenolic content and reducing potential was observed, indicating that phenolic compounds and reducers present in extract are major contributors to the antioxidant potential. Thus, this plant extract could be used as a potent natural antioxidant.

Microstructure and homogeneity of distribution of mineralised struts determine callus strength.

Non-invasive assessment of fracture healing, both in clinical and animal studies, has gained favour as surrogate measure to estimate regain of mechanical function. Micro-computed tomography (µCT) parameters such as fracture callus volume and mineralisation have been used to estimate callus mechanical competence. However, no in-depth information has been reported on microstructural parameters in estimating callus mechanical competence. The goal of this study is to use differently conditioned mice exhibiting good and impaired fracture healing outcomes and investigate the relationship between µCT imaging parameters (volume, mineralisation, and microstructure) that best estimate the callus strength and stiffness as it develops over time. A total of 99 mice with femoral fracture and intramedullary stabilisation were divided into four groups according to conditioning: wild type, NF1 knock-out, RAG1 knock-out and macrophage depleted. Animals were sacrificed at 14, 21, 28 or 35 days and µCT parameters and torsional stiffness and strength were assessed post-sacrifice. Using linear regression for all groups and time points together, torsional stiffness could be estimated with strut thickness, strut number and strut homogeneity (R² = 0.546, p < 0.0001); torsional strength could be estimated using bone mineral density, strut thickness and strut homogeneity (R² = 0.568, p < 0.0001). Differently conditioned mice that result in different fracture healing outcomes have been shown to result in varying structural, material and volumetric µCT parameters which can be used to estimate regain of bone strength. This study is the first to demonstrate that microstructure and strut homogeneity influence callus stiffness and strength.

Periosteal cells are a major source of soft callus in bone fracture.

During the healing process after bone fracture, soft callus forms adjacent to the fracture site, is replaced by hard callus, and is finally remodeled to the original bone configuration. Although the cambium layer of the periosteum is reported to play an essential role in callus formation, we still lack direct in vivo evidence of this. To investigate the cell lineage of the soft callus, we analyzed the process of fracture healing in Prx1-Cre;ROSA26 reporter (R26R), Col1a1(3.6 kb)-Cre;R26R, Col1a1(2.3 kb)-Cre;R26R, Sox9-CreERT2;R26R, and Sox9-LacZ mice with X-gal staining. In the Prx1-Cre;R26R, in which the cells of the periosteum stained for X-gal before fracture, all cells in the soft callus were X-gal positive, whereas in the Col1a1(3.6 kb)-Cre;R26R mice, the cells in the periosteum before fracture stained for X-gal and the soft callus was partly composed of X-gal-positive cells. In contrast, in the Col1a1(2.3 kb)-Cre;R26R mice, in which the mature osteoblasts in the cambium layer of the periosteum were marked before fracture, no cells in the soft callus at the fracture site were X-gal positive. These results suggest that most of the cells in the soft callus are derived from the mesenchymal progenitors in the periosteum, and not from mature osteoblastic cells. Interestingly, in the Sox9-LacZ mice, Sox9-expressing X-gal-positive cells emerged in the periosteum adjacent to the fracture site 3 days after fracture. We demonstrated this by injecting tamoxifen into the Sox9-CreERT2;R26R mice for 3 days after fracture, so that these Sox9-expressing periosteal cells gave rise to cells in the soft and hard calli. Our findings show that the periosteal cells in which Sox9 expression is induced just after fracture are the major source of the chondrocytes and osteoblasts in the fracture callus.

Systemic inflammation induced by a thoracic trauma alters the cellular composition of the early fracture callus.

BACKGROUND: We recently demonstrated that a blunt chest trauma, a strong inducer of the posttraumatic systemic inflammatory response and one of the most critical injuries in polytrauma patients, significantly delayed fracture healing in rats, possibly by the interaction of the systemic inflammation with early regeneration processes locally at the fracture site. The underlying cellular mechanisms, however, have as yet remained unknown. Therefore, the aim of this study was to analyze the cellular and morphologic composition of the early fracture callus after a blunt chest trauma. METHODS: Rats received an osteotomy of the right femur stabilized by an external fixator in combination with a blunt chest trauma or not. The animals were killed after 3, 7, and 35 days, and the fracture calli were analyzed histologically for new tissue formation, polymorphonuclear leucocytes, macrophages, osteoclasts, and the presence of the proinflammatory cytokine interleukin 6. RESULTS: The blunt chest trauma considerably increased the number of polymorphonuclear leucocytes in the callus by Day 3 compared with animals with isolated fractures. The number of macrophages was significantly reduced by the thoracic trauma at Days 3 and 7. The number of osteoclasts was not changed at any postoperative time point. After 3 days, the blunt chest trauma led to a significantly stronger interleukin 6 staining within the periosteal callus in zones of intramembranous ossification. During the time of cortical bridging at Day 35, the amount of newly formed bone was significantly decreased after blunt chest trauma. CONCLUSION: Our results suggest that the systemic posttraumatic inflammation induced by a thoracic trauma disturbed the inflammatory balance during the early healing stage by altering the recruitment of inflammatory cells and cytokine expression locally at the fracture site and thus impaired fracture healing. These findings provide new insights in the pathomechanisms of impaired fracture healing in patients experiencing severe trauma.

Bone marrow-engrafted cells after mice umbilical cord blood transplantation differentiate into osteoblastic cells in response to fracture and placement of titanium screws.

As the in vivo function of bone marrow-engrafted umbilical cord blood (UCB)-derived mesenchymal cells (UCBCs) after UCB transplantation is unknown, we examined in vivo osteoblastic differentiation using mouse UCB transplantation and fracture models. UCBCs obtained from GFP transgenic mice were intravenously injected into irradiated C57BL/6 mice. After three months, the in vivo osteoblastic differentiation potential of bone marrow-engrafted UCBCs was examined histologically using a mouse fracture model. GFP-positive UCBCs were detected in the bone marrow of recipient mice. On day 7, UCBCs were observed in the fracture gap and surrounding the titanium screws of the fixation device. The UCBCs were also positive for alkaline phosphatase and von Kossa staining. By day 14, UCBCs were observed around and within a formed intramedullary callus. The newly formed woven bone consisted of ALP- and von Kossa-positive cells. Our findings suggest that UCBCs contribute to the fracture healing process after bone marrow engraftment and that UCBC transplantation can fully reconstruct not only hematopoietic cells but also mesenchymal cell lineages.