Model Of Bone Injury Research Articles

An ALK2 inhibitor, BLU-782, prevents heterotopic ossification in a mouse model of fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease driven by gain-of-function variants in activin receptor-like kinase 2 (ALK2), the most common variant being ALK2R206H. In FOP, ALK2 variants display increased and dysregulated signaling through the bone morphogenetic protein (BMP) pathway resulting in progressive and permanent replacement of skeletal muscle and connective tissues with heterotopic bone, ultimately leading to severe debilitation and premature death. Here, we describe the discovery of BLU-782 (IPN60130), a small-molecule ALK2R206H inhibitor developed for the treatment of FOP. A small-molecule library was screened in a biochemical ALK2 binding assay to identify potent ALK2 binding compounds. Iterative rounds of structure-guided drug design were used to optimize compounds for ALK2R206H binding, ALK2 selectivity, and other desirable pharmacokinetic properties. BLU-782 preferentially bound to ALK2R206H with high affinity, inhibiting signaling from ALK2R206H and other rare FOP variants in cells in vitro without affecting signaling of closely related homologs ALK1, ALK3, and ALK6. In vivo efficacy of BLU-782 was demonstrated using a conditional knock-in ALK2R206H mouse model, where prophylactic oral dosing reduced edema and prevented cartilage and heterotopic ossification (HO) in both muscle and bone injury models. BLU-782 treatment preserved the normal muscle-healing response in ALK2R206H mice. Delayed dosing revealed a short 2-day window after injury when BLU-782 treatment prevented HO in ALK2R206H mice, but dosing delays of 4 days or longer abrogated HO prevention. Together, these data suggest that BLU-782 may be a candidate for prevention of HO in FOP.

Novel 3D-printing bilayer GelMA-based hydrogel containing BP, β-TCP and exosomes for cartilage–bone integrated repair

The integrated repair of cartilage and bone involves the migration and differentiation of cells, which has always been a difficult problem to be solved. We utilize the natural biomaterial gelatin to construct gelatin methacryloyl (GelMA), a hydrogel scaffold with high cell affinity. GelMA is mixed with different components to print a bi-layer porous hydrogel scaffold with different modulus and composition in upper and lower layers through three-dimensional (3D) printing technology. The upper scaffold adds black phosphorus (BP) and human umbilical cord mesenchymal stem cells (hUMSCs) exosomes (exos) in GelMA, which has a relatively lower elastic modulus and is conducive to the differentiation of BMSCs into cartilage. In the lower scaffold, in addition to BP and hUMSCs exos, β-tricalcium phosphate (β-TCP), which has osteoconductive and osteoinductive effects, is added to GelMA. The addition of β-TCP significantly enhances the elastic modulus of the hydrogel scaffold, which is conducive to the osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs). In vitro experiments have confirmed that the bi-layer scaffolds can promote osteogenesis and chondrogenic differentiation respectively. And in the rabbit cartilage–bone injury model, MRI and micro-CT results show that the 3D printed bi-layer GelMA composite scaffold has a repair effect close to normal tissue.

Exogenously delivered iPSCs disrupt the natural repair response of endogenous MPCs after bone injury

Promoting bone healing including fracture non-unions are promising targets for bone tissue engineering due to the limited success of current clinical treatment methods. There has been significant research on the use of stem cells with and without biomaterial scaffolds to treat bone fractures due to their promising regenerative capabilities. However, the relative roles of exogenous vs. endogenous stem cells and their overall contribution to in vivo fracture repair is not well understood. The purpose of this study was to determine the interaction between exogenous and endogenous stem cells during bone healing. This study was conducted using a standardized burr-hole bone injury model in a mesenchymal progenitor cell (MPC) lineage-tracing mouse under normal homeostatic and osteoporotic conditions. Burr-hole injuries were treated with a collagen-I biomaterial loaded with and without labelled induced pluripotent stem cells (iPSCs). Using lineage-tracing, the roles of exogenous and endogenous stem cells during bone healing were examined. It was observed that treatment with iPSCs resulted in muted healing compared to untreated controls in intact mice post-injury. When the cell populations were examined histologically, iPSC-treated burr-hole defects presented with a dramatic reduction in endogenous MPCs and cell proliferation throughout the injury site. However, when the ovaries were removed and an osteoporotic-like phenotype induced in the mice, iPSCs treatment resulted in increased bone formation relative to untreated controls. In the absence of iPSCs, endogenous MPCs demonstrated robust proliferative and osteogenic capacity to undertake repair and this behaviour was disrupted in the presence of iPSCs which instead took on an osteoblast fate but with little proliferation. This study clearly demonstrates that exogenously delivered cell populations can impact the normal function of endogenous stem/progenitor populations during the normal healing cascade. These interactions need to be better understood to inform cell and biomaterial therapies to treat fractures.

Cadmium accelerates autophagy of osteocytes by inhibiting the PI3K/AKT/mTOR signaling pathway.

Cadmium (Cd) can damage bone cells and cause osteoporosis. Osteocytes are the most numerous bone cells and also important target cells for Cd-induced osteotoxic damage. Autophagy plays important role in the progression of osteoporosis. However, osteocyte autophagy in Cd-induced bone injury is not well characterized. Thus, we established a Cd-induced bone injury model in BALB/c mice and a cellular damage model in MLO-Y4 cells. Aqueous Cd exposure for 16 months showed an increase in plasma alkaline phosphatase (ALP) activity and increase in urine calcium (Ca) and phosphorus (P) concentrations in vivo. Moreover, expression level of autophagy-related microtubule-associated protein 1A/1B-light chain 3 II (LC3II) and autophagy-related 5 (ATG5) proteins were induced, and the expression of sequestosome-1 (p62) was reduced, along with Cd-induced trabecular bone damage. In addition, Cd inhibited the phosphorylation of mammalian target of rapamycin (mTOR), protein kinase B (AKT), and phosphatidylinositol 3-kinase (PI3K). In vitro, 80 μM Cd concentrations exposure upregulated LC3II protein expression, and downregulated of p62 protein expression. Similarly, we found that treatment with 80 μM Cd resulted in a reduction in the phosphorylation levels of mTOR, AKT, and PI3K. Further experiments revealed that addition of rapamycin, an autophagy inducer, enhanced autophagy and alleviated the Cd-induced damage to MLO-Y4 cells. The findings of our study reveal for the first time that Cd causes damage to both bone and osteocytes, as well as induces autophagy in osteocytes and inhibits PI3K/AKT/mTOR signaling, which could be a protective mechanism against Cd-induced bone injury.

Sequential application of small molecule therapy enhances chondrogenesis and angiogenesis in murine segmental defect bone repair.

The increasing incidence of physiologic/pathologic conditions that impair the otherwise routine healing of endochondral bone fractures and the occurrence of severe bone injuries necessitate novel approaches to enhance clinically challenging bone fracture repair. To promote the healing of nonunion fractures, we tested an approach that used two small molecules to sequentially enhance cartilage development and conversion to the bone in the callus of a murine femoral segmental defect nonunion model of bone injury. Systemic injections of smoothened agonist 21k(SAG21k) were used to stimulate chondrogenesis through the activation of the sonic hedgehog (SHH) pathway early in bone repair, while injections of the prolyl hydroxylase domain (PHD)2 inhibitor, IOX2, were used to stimulate hypoxia signaling-mediated endochondral bone formation. The expression of SHH pathway genes and Phd2 target genes was increased in chondrocyte cell lines in response to SAG21k and IOX2 treatment, respectively. The segmental defect responded to sequential systemic administration of these small molecules with increased chondrocyte expression of PTCH1, GLI1, and SOX9 in response to SAG and increased expression of hypoxia-induced factor-1α and vascular endothelial growth factor-A in the defect tissues in response to IOX2. At 6 weeks postsurgery, the combined SAG-IOX2 therapy produced increased bone formation in the defect with the bony union over the injury.Clinical significance: This therapeutic approach was successful in promoting cartilage and bone formation within a critical-size segmental defect and established the utility of a sequential small molecule therapy for the enhancement of fracture callus development in clinically challenging bone injuries.

REGENERATIVE EFFECTS OF MOUSE ADIPOSE-DERIVED MULTIPOTENT STROMAL CELLS IN A MICROMASS GRAFT FOR THE TREATMENT OF BONE INJURY MODEL

Background. Adipose-derived stem cells (ADSCs) are a promising source for the regeneration of bone tissue injuries. At the same time, three-dimensional cultures provide spatial organization of stem cells for optimal intercellular signaling, contact interaction and increase the efficiency of directed osteogenic differentiation prior to further transplantation.
 The aim of the study was to establish the regenerative potential of mouse adipose-derived stem cells in micromass grafts differentiated into the osteogenic direction to restore the bone injury in mice.
 Methods. Three-dimensional micromass cultures of murine ADSCs with further differentiation into osteogenic direction were obtained. The migration potential of cells from micromass in vitro and the effectiveness of differentiation by staining for alkaline phosphatase were evaluated. Mice with the model of femoral bone injury were transplanted with ADSCs micromass grafts and 21 days later the lesion site was examined by histological and morphometric methods.
 Results. The protocols for the cultivation and directed osteogenic differentiation of ADSCs in the three-dimensional micromass culture have been developed. Alkaline phosphatase production was demonstrated in cells that migrated from micromass, confirming the effectiveness of differentiation. In macroscopic examination 21 days after graft transplantation, the defect sites in femur were filled with dense tissue, while in control bones without the use of transplants, the size of the defect by 80 ± 6 % corresponded to the initial diameter and depth of injury. Histological examination of femoral bone lesions in the area of transplantation of micromass grafts revealed the formation of granulation tissue followed by the replacement of defects with newly formed bone tissue with thickening of periosteum and compact bone substance, similar to callus in fracture regeneration. In animals that underwent transplantation of micromass without prior osteogenic differentiation, the diameter of the zone of active regeneration of the diaphysis at the site of injury was 1.3 ± 0.2 mm while in the group with transplantation of directed differentiated graft it was significantly lower (0.37 ± 0.12 mm, p ≤ 0.05).
 Conclusions. Three-dimensional grafts of adipose-derived multipotent mesenchymal stromal cells cultured in micromass are able to improve bone tissue regeneration in a model of bone injury in mice. In this case, the grafts differentiated into osteogenic direction, provide better morphological indicators of bone recovery, compared with the micromass without prior differentiation.

MODELING OF BONE INJURIES IN ANIMAL EXPERIMENTS

In this review, we discuss the advantages and disadvantages of the existing bone defect animal models and tissue engineering techniques applied in studying novel bone defect regenerative approaches. The paper suggests the requirements for an optimal animal model, as well as analyzes in vivo bone injury models widely used in testing. The authors briefly review the methods of experimentally produced lesions of long bones, calvarial bones, mandibular bones in different animals. This review also describes the standardization techniques allowing one to evaluate the process of osteogenesis and bone-implant interactions. That will help researchers thoroughly plan and conduct experiments according to the bioethical principles.

Animal Experimental Study on Repair of Alveolar Bone Defect with Double-Pipe Biphase Bioceramic Polyetheretherketone Composite

Polyetheretherketone (PEEK) has the advantages of good biocompatibility, thus become a widely used bone remodeling material. Bioceramics are also highly effective bone repair materials. However, the repairing effect of biphasic bioceramics combined with polyetheretherketone composites on alveolar bone defects has not been elucidated. SD rats were separated into control group that was established as alveolar bone injury model; PEEK group that the alveolar bone injury model was repaired with polyether ether ketone; and composite group that alveolar bone injury model was repaired with double-tube biphase bioceramic/polyetheretherketone. After 8 weeks of treatment, the bone mineral density (BMD) changes were assessed by X-ray absorptiometry. HE staining was used to analyze the changes of tooth defect. The change of alkaline phosphatase (ALP) content was analyzed. Real-time PCR was performed to measure osteogenic factors Opn and Runx2 expressions. Serum BMP-2 level was analyzed by ELISA. The alveolar ridge height was compared and the alveolar bone repair rate was calculated. In the PEEK group and the composite group, BMD was significantly increased, bone was repaired, Runx2 and Opn mRNA expression was upregulated and ALP activity was enhanced along with elevated BMP-2 secretion, alveolar ridge height and bone repair rate compared to control group (P < 0 05). The composite group exhibited more significant changes compared with PEEK group (P < 0 05). Double-tube biphasic bioceramic/polyetheretherketone composites can repair alveolar bone defects, promote osteogenic differentiation, induce BMP-2 secretion, enhance bone density, and accelerate alveolar bone repair.

A segmental defect adaptation of the mouse closed femur fracture model for the analysis of severely impaired bone healing.

ObjectiveTo better characterize nonunion endochondral bone healing and evaluate novel therapeutic approaches for critical size defect healing in clinically challenging bone repair, a segmental defect model of bone injury was adapted from the three‐point bending closed fracture technique in the murine femur.MethodsThe mouse femur was surgically stabilized with an intramedullary threaded rod with plastic spacers and the defect adjusted to different sizes. Healing of the different defects was analyzed by radiology and histology to 8 weeks postsurgery. To determine whether this model was effective for evaluating the benefits of molecular therapy, BMP‐2 was applied to the defect and healing then examined.ResultsIntramedullary spacers were effective in maintaining the defect. Callus bone formation was initiated but was arrested at defect sizes of 2.5 mm and above, with no more progress in callus bone development evident to 8 weeks healing. Cartilage development in a critical size defect attenuated very early in healing without bone development, in contrast to the closed femur fracture healing, where callus cartilage was replaced by bone. BMP‐2 therapy promoted osteogenesis of the resident cells of the defect, but there was no further callus development to indicate that healing to pre‐surgery bone structure was successful.ConclusionsThis segmental defect adaptation of the closed femur fracture model of murine bone repair severely impairs callus development and bone healing, reflecting a challenging bone injury. It is adjustable and can be compared to the closed fracture model to ascertain healing deficiencies and the efficacy of therapeutic approaches.

MGF-19E peptide promoted proliferation, differentiation and mineralization of MC3T3-E1 cell and promoted bone defect healing.

The repair of segmental bone defects and bone fractures is a clinical challenge involving high risk and postsurgical morbidity. Bone injury and partial bone tumor resection via traditional bone grafting result in high complications. Growth factors have been proposed as alternatives to promote bone repair and formation and circumvent these limitations. In this study, we classified different lengths of mechano growth factor (MGF) E peptides in different species and analyzed their effects on MC3T3-E1 cell proliferation, cell cycle, alkaline phosphatase (ALP) activity, differentiation-related factor expression, and cell mineralization. A rabbit bone injury model was constructed, and the repair function of MGF E peptide was verified by injecting the candidate MGF E peptide. We analyzed 52 different MGF-E peptides and classified them into the following four categories: T-MGF-25E, M-MGF-25E, T-MGF-19E, and M-MGF-19E. These peptides were synthesized for further study. T-MGF-19E peptide obviously promoted cell proliferation by regulating cell cycle after MGF E peptide treatment at 72h. T-MGF-25E and T-MGF-19E peptide significantly promoted the differentiation of osteoblasts on day 14, and M-MGF-25E peptide promoted cell differentiation on day 7. T-MGF-19E, T-MGF-25E, and M-MGF-19E significantly promoted osteoblast mineralization, with T-MGF19E showing the most significant effect. These results implied that T-MGF19E peptide could remarkably promote MC3T3-E1 cell proliferation, differentiation, and mineralization. The rabbit bone defect model showed that the low-dose T-MGF-19E peptide significantly promoted bone injury healing, suggesting its promoting effect on the healing of bone injury.

Low-intensity Pulsed Ultrasound regulates alveolar bone homeostasis in experimental Periodontitis by diminishing Oxidative Stress.

Periodontitis is a widespread oral disease that results in the loss of alveolar bone. Low-intensity pulsed ultrasound (LIPUS), which is a new therapeutic option, promotes alveolar bone regeneration in periodontal bone injury models. This study investigated the protective effect of LIPUS on oxidative stress in periodontitis and the mechanism underlying this process.Methods: An experimental periodontitis model was induced by administering a ligature. Immunohistochemistry was performed to detect the expression levels of oxidative stress, osteogenic, and osteoclastogenic markers in vivo. Cell viability and osteogenic differentiation were analyzed using the Cell Counting Kit-8, alkaline phosphatase, and Alizarin Red staining assays. A reactive oxygen species assay kit, lipid peroxidation MDA assay kit, and western blotting were used to determine oxidative stress status in vitro. To verify the role of nuclear factor erythroid 2-related factor 2 (Nrf2), an oxidative regulator, during LIPUS treatment, the siRNA technique and Nrf2-/- mice were used. The PI3K/Akt inhibitor LY294002 was utilized to identify the effects of the PI3K-Akt/Nrf2 signaling pathway.Results: Alveolar bone resorption, which was experimentally induced by periodontitis in vivo, was alleviated by LIPUS via activation of Nrf2. Oxidative stress, induced via H2O2 treatment in vitro, inhibited cell viability and suppressed osteogenic differentiation. These effects were also alleviated by LIPUS treatment via Nrf2 activation. Nrf2 silencing blocked the antioxidant effect of LIPUS by diminishing heme oxygenase-1 expression. Nrf2-/- mice were susceptible to ligature-induced periodontitis, and the protective effect of LIPUS on alveolar bone dysfunction was weaker in these mice. Activation of Nrf2 by LIPUS was accompanied by activation of the PI3K/Akt pathway. The oxidative defense function of LIPUS was inhibited by exposure to LY294002 in vitro.Conclusions: These results demonstrated that LIPUS regulates alveolar bone homeostasis in periodontitis by attenuating oxidative stress via the regulation of PI3K-Akt/Nrf2 signaling. Thus, Nrf2 plays a pivotal role in the protective effect exerted by LIPUS against ligature-induced experimental periodontitis.

Zebrafish Models of Human Skeletal Disorders: Embryo and Adult Swimming Together.

Danio rerio (zebrafish) is an elective model organism for the study of vertebrate development because of its high degree of homology with human genes and organs, including bone. Zebrafish embryos, because of the optical clarity, small size, and fast development, can be easily used in large-scale mutagenesis experiments to isolate mutants with developmental skeletal defects and in high-throughput screenings to find new chemical compounds for the ability to revert the pathological phenotype. On the other hand, the adult zebrafish represents another powerful resource for pathogenic and therapeutic studies about adult human bone diseases. In fish, some characteristics such as bone turnover, reparation, and remodeling of the adult bone tissue cannot be found at the embryonic stage. Several pathological models have been established in adult zebrafish such as bone injury models, osteoporosis, and genetic diseases such as osteogenesis imperfecta. Given the growing interest for metabolic diseases and their complications, adult zebrafish models of type 2 diabetes and obesity have been recently generated and analyzed for bone complications using scales as model system. Interestingly, an osteoporosis-like phenotype has been found to be associated with metabolic alterations suggesting that bone complications share the same mechanisms in humans and fish. Embryo and adult represent powerful resources in rapid development to study bone physiology and pathology from different points of view.

VEGFA From Early Osteoblast Lineage Cells (Osterix+) Is Required in Mice for Fracture Healing.

Bone formation via intramembranous and endochondral ossification is necessary for successful healing after a wide range of bone injuries. The pleiotropic cytokine, vascular endothelial growth factor A (VEGFA) has been shown, via nonspecific pharmacologic inhibition, to be indispensable for angiogenesis and ossification following bone fracture and cortical defect repair. However, the importance of VEGFA expression by different cell types during bone healing is not well understood. We sought to determine the role of VEGFA from different osteoblast cell subsets following clinically relevant models of bone fracture and cortical defect. Ubiquitin C (UBC), Osterix (Osx), or Dentin matrix protein 1 (Dmp1) Cre-ERT2 mice (male and female) containing floxed VEGFA alleles (VEGFAfl/fl ) were either given a femur full fracture, ulna stress fracture, or tibia cortical defect at 12 weeks of age. All mice received tamoxifen continuously starting 2 weeks before bone injury and throughout healing. UBC Cre-ERT2 VEGFAfl/fl (UBC cKO) mice, which were used to mimic nonspecific inhibition, had minimal bone formation and impaired angiogenesis across all bone injury models. UBC cKO mice also exhibited impaired periosteal cell proliferation during full fracture, but not stress fracture repair. Osx Cre-ERT2 VEGFAfl/fl (Osx cKO) mice, but not Dmp1 Cre-ERT2 VEGFAfl/fl (Dmp1 cKO) mice, showed impaired periosteal bone formation and angiogenesis in models of full fracture and stress fracture. Neither Osx cKO nor Dmp1 cKO mice demonstrated significant impairments in intramedullary bone formation and angiogenesis following cortical defect. These data suggest that VEGFA from early osteolineage cells (Osx+), but not mature osteoblasts/osteocytes (Dmp1+), is critical at the time of bone injury for rapid periosteal angiogenesis and woven bone formation during fracture repair. Whereas VEGFA from another cell source, not from the osteoblast cell lineage, is necessary at the time of injury for maximum cortical defect intramedullary angiogenesis and osteogenesis. © 2019 American Society for Bone and Mineral Research.

Evaluation of a Critical Sized Bone Defect in a Porcine Tibial Model

Background/Hypothesis:
 Few large animal bone injury models exist. We present a porcine tibial model of metacritical and critical sized bone defects for the simulation of traumatic bone injuries.
 Project Methods:
 16 pigs were used in this study. The pigs were divided into 3 groups (n=4-8/group). In 12 pigs, a 25 mm osteotomy was created in the tibia and the space was filled with a 25 mm scaffold. 8 of the 25 mm scaffolds were secured with an intramedullary (IM) nail and 4 of the scaffolds were secured with plates. In 4 pigs a 40 mm osteotomy was created, filled with a 40 mm scaffold, and secured with plates. Fracture healing was assessed 3 months post-operatively using the Radiographic Union Score for Tibial Fractures (RUST) criteria.
 Results:
 For the 25 mm IM group, none of the 8 pigs achieved cortical bone union at 3 months post-op. In contrast, cortical union was observed in all of the 25 mm defects secured with plates. None of the 40 mm defects secured with plates achieved cortical union.
 Conclusion/Potential Impact:
 The failure of the 25 mm defect secured with an IM nail and the 40 mm defect secured with plates demonstrates that these are critical sized defect models. However, because the 25 mm defect secured with bridge plates did heal, we have termed this a metacritical sized defect. Identification of a defect size that can heal or not heal based on the fixation technique is a powerful translational model for testing therapies.

Translation of remote control regenerative technologies for bone repair

The role of biomechanical stimuli, or mechanotransduction, in normal bone homeostasis and repair is understood to facilitate effective osteogenesis of mesenchymal stem cells (MSCs) in vitro. Mechanotransduction has been integrated into a multitude of in vitro bone tissue engineering strategies and provides an effective means of controlling cell behaviour towards therapeutic outcomes. However, the delivery of mechanical stimuli to exogenous MSC populations, post implantation, poses a significant translational hurdle. Here, we describe an innovative bio-magnetic strategy, MICA, where magnetic nanoparticles (MNPs) are used to remotely deliver mechanical stimuli to the mechano-receptor, TREK-1, resulting in activation and downstream signalling via an external magnetic array. In these studies, we have translated MICA to a pre-clinical ovine model of bone injury to evaluate functional bone repair. We describe the development of a magnetic array capable of in vivo MNP manipulation and subsequent osteogenesis at equivalent field strengths in vitro. We further demonstrate that the viability of MICA-activated MSCs in vivo is unaffected 48 h post implantation. We present evidence to support early accelerated repair and preliminary enhanced bone growth in MICA-activated defects within individuals compared to internal controls. The variability in donor responses to MICA-activation was evaluated in vitro revealing that donors with poor osteogenic potential were most improved by MICA-activation. Our results demonstrate a clear relationship between responders to MICA in vitro and in vivo. These unique experiments offer exciting clinical applications for cell-based therapies as a practical in vivo source of dynamic loading, in real-time, in the absence of pharmacological agents.

* Bone Regeneration Mediated by a Bioactive and Biodegradable Extracellular Matrix-Like Hydrogel Based on Elastin-Like Recombinamers.

The morbidity of bone fractures and defects is steadily increasing due to changes in the age pyramid. As such, novel biomaterials that are able to promote the healing and regeneration of injured bones are needed to overcome the limitations of auto-, allo-, and xenografts, while providing a ready-to-use product that may help to minimize surgical invasiveness and duration. In this regard, recombinant biomaterials, such as elastin-like recombinamers (ELRs), are very promising as their design can be tailored by genetic engineering, thus allowing scalable production and batch-to-batch consistency, among others. Furthermore, they can self-assemble into physically crosslinked hydrogels above a certain transition temperature, in this case body temperature, but are injectable below this temperature, thereby markedly reducing surgical invasiveness. In this study, we have developed two bioactive hydrogel-forming ELRs, one including the osteogenic and osteoinductive bone morphogenetic protein-2 (BMP-2) and the other the Arg-Gly-Asp (RGD) cell adhesion motif. The combination of these two novel ELRs results in a BMP-2-loaded extracellular matrix-like hydrogel. Moreover, elastase-sensitive domains were included in both ELR molecules, thereby conferring biodegradation as a result of enzymatic cleavage and avoiding the need for scaffold removal after bone regeneration. Both ELRs and their combination showed excellent cytocompatibility, and the culture of cells on RGD-containing ELRs resulted in optimal cell adhesion. In addition, hydrogels based on a mixture of both ELRs were implanted in a pilot study involving a femoral bone injury model in New Zealand white rabbits, showing complete regeneration in six out of seven cases, with the other showing partial closure of the defect. Moreover, bone neoformation was confirmed using different techniques, such as radiography, computed tomography, and histology. This hydrogel system therefore displays significant potential in the regeneration of bone defects, promoting self-regeneration by the surrounding tissue with no involvement of stem cells or osteogenic factors other than BMP-2, which is released in a controlled manner by elastase-mediated cleavage from the ELR backbone.

Resting and injury-induced inflamed periosteum contain multiple macrophage subsets that are located at sites of bone growth and regeneration.

Better understanding of bone growth and regeneration mechanisms within periosteal tissues will improve understanding of bone physiology and pathology. Macrophage contributions to bone biology and repair have been established but specific investigation of periosteal macrophages has not been undertaken. We used an immunohistochemistry approach to characterize macrophages in growing murine bone and within activated periosteum induced in a mouse model of bone injury. Osteal tissue macrophages (osteomacs) and resident macrophages were distributed throughout resting periosteum. In tissues collected from 4-week-old mice, osteomacs were observed intimately associated with sites of periosteal diaphyseal and metaphyseal bone dynamics associated with normal growth. This included F4/80+Mac-2-/low osteomac association with extended tracks of bone formation (modeling) on diphyseal periosteal surfaces. Although this recapitulated endosteal osteomac characteristics, there was subtle variance in the morphology and spatial organization of periosteal modeling-associated osteomacs, which likely reflects the greater structural complexity of periosteum. Osteomacs, resident macrophages and inflammatory macrophages (F4/80+Mac-2hi) were associated with the complex bone dynamics occurring within the periosteum at the metaphyseal corticalization zone. These three macrophage subsets were also present within activated native periosteum after bone injury across a 9-day time course that spanned the inflammatory through remodeling bone healing phases. This included osteomac association with foci of endochondral ossification within the activated native periosteum. These observations confirm that osteomacs are key components of both osteal tissues, in spite of salient differences between endosteal and periosteal structure and that multiple macrophage subsets are involved in periosteal bone dynamics.

A single administration of dexamethasone may protect tibial plateau cartilage in a mechanically stable surgical model of post traumatic osteoarthritis

Purpose: An acute inflammatory phase follows an injury to the knee joint and initiates a cascade of biochemical events that can contribute to degeneration of the cartilage. Cartilage degeneration after injury is central in the development of posttraumatic osteoarthritis (PTOA) – a debilitating disease that is increasingly affecting active younger individuals. It is currently believed that inflammation following intra-articular bleeding from joint injuries could be a cause in the development of PTOA. To study the effects of inflammation alone within the joint, an injury model was developed in which tissue structures responsible for joint function and stability were not directly impacted. Preliminary evidence in this rabbit model of intra-articular bone injury, intended to simulate posttraumatic hemarthrosis, has shown what appears to be an acute inflammatory phase that resolves after surgery and yet still results in the development of significant PTOA. Therefore, we hypothesize that if insult to the joint increases the expression level of acute inflammatory mediators that expose the cartilage to injury then an intervention that inhibits acute inflammation (dexamethasone) will have positive outcomes on cartilage health within this rabbit bone injury model. Dexamethasone (dex) is a glucocorticoid with potent anti-inflammatory properties that has been widely adopted for clinical use (e.g. in the treatment of rheumatoid arthritis, asthma, and various cancers). Methods: Skeletally mature adult female rabbits aged 52 weeks (n = 18) were used in this study. Six animals were not subjected to any interventions and served as un-operated controls. The remaining 12 were all subjected to a femoral notch drill surgery following the guidelines of our institutional animal care facility. Of this group, six rabbits were treated with one intra-articular injection of Dex (0.5 mg/kg) at the time of surgery. As surgical controls, the remaining 6 animals were subjected to drill surgery, but did not receive any dexamethasone treatment. Animals were sacrificed at 9 weeks post surgery. Histological assessment was completed by two blinded observers using a modified Mankin protocol. qPCR was used to analyze the mRNA levels for select anabolic and catabolic molecules, and values were normalized to 18S mRNA. Kruskal-Wallis with Dunn’s multiple comparisons test were used to investigate treatment group differences (significance accepted at p ≤ 0.05). Results: Histological evaluations (Figure 1A) revealed that at 9 weeks post surgery the cartilage on the tibial plateaus of the drill animals was significantly more damaged than that of the un-operated controls and the animals that received the dex treatment after injury. There were no significant differences between the tibial plateau cartilage of the normal and drill + dex treatment groups, suggesting a positive role for dexamethasone. Furthermore, the mRNA analysis (Figure 1B) revealed that even a single dose of dexamethasone at the time of surgery, was sufficient to attenuate the expression of degradative proteinase matrix metalloproteinase 3 (MMP-3), proteoglycans such as versican and biglycan as well as anabolic marker collagen type II (Col-II) in tibial plateau cartilage. Conclusions: Even in the absence of severe biomechanical changes, the cartilage of the tibial plateau is subject to degeneration in this model. This strongly suggests that initial biological changes are sufficient to drive many of the changes observed within the joint. The results of this study suggest that Dexamethasone treatment given at the time of drill surgery had a positive impact in reducing the degradation of the cartilage covering the tibial plateau. Interestingly, treatment with this steroid attenuated the mRNA levels for both anabolic and catabolic molecules.

Acceleration of Bone Repair in NOD/SCID Mice by Human Monoosteophils, Novel LL-37-Activated Monocytes

BackgroundAn incomplete understanding of bone forming cells during wound healing and ectopic calcification has led to a search for circulating cells that may fulfill this function. Previously, we showed that monoosteophils, a novel lineage of calcifying/bone-forming cells generated by treatment of monocytes with the natural peptide LL-37, are candidates. In this study, we have analyzed their gene expression profile and bone repair function.Methods and FindingsHuman monoosteophils can be distinguished from monocytes, macrophages and osteoclasts by their unique up-regulation of integrin α3 and down-regulation of CD14 and CD16. Monoosteophils express high mRNA and protein levels of SPP1 (osteopontin), GPNMB (osteoactivin), CHI3L1 (cartilage glycoprotein-39), CHIT1 (Chitinase 1), MMP-7, CCL22 and MAPK13 (p38MAPKδ). Monocytes from wild type, but not MAPK13 KO mice are also capable of monoosteophil differentiation, suggesting that MAPK13 regulates this process. When human monoosteophils were implanted in a freshly drilled hole in mid-diaphyseal femurs of NOD/SCID mice, significant bone repair required only 14 days compared to at least 24 days in control treated injuries.ConclusionHuman derived monoosteophils, characterized as CD45+α3+α3β+CD34−CD14−BAP (bone alkaline phosphatase)− cells, can function in an animal model of bone injury.

Assessment of the efficacy of MRI for detection of changes in bone morphology in a mouse model of bone injury

To determine whether magnetic resonance imaging (MRI) could be used to track changes in skeletal morphology during bone healing using high-resolution micro-computed tomography (μCT) as a standard. We used a mouse model of bone injury to compare μCT with MRI. Surgery was performed to induce a burr hole fracture in the mouse tibia. A selection of biomaterials was immediately implanted into the fractures. First we optimized the imaging sequences by testing different MRI pulse sequences. Then changes in bone morphology over the course of fracture repair were assessed using in vivo MRI and μCT. Histology was performed to validate the imaging outcomes. The rapid acquisition with relaxation enhancement (RARE) sequence provided sufficient contrast between bone and the surrounding tissues to clearly reveal the fracture. It allowed detection of the fracture clearly 1 and 14 days postsurgery and revealed soft tissue changes that were not clear on μCT. In MRI and μCT the fracture was seen at day 1 and partial healing was detected at day 14. The RARE sequence was the most suitable for MRI bone imaging. It enabled the detection of hard and even soft tissue changes. These findings suggest that MRI could be an effective imaging modality for assessing changes in bone morphology and pathobiology.