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A soluble form of BMP receptor type 1A (mBMPR1A-mFC) acts as an antagonist to endogenous BMPR1A and has been shown to increase bone mass in mice. The goal of this study was to examine the effects of mBMPR1A-mFC on secondary fracture healing. Treatment consisted of 10 mg/kg intraperitoneal injections of mBMPR1A-mFC twice weekly in male C57BL/6 mice. Treatment beginning at 1, 14, and 21 days post-fracture assessed receptor function during endochondral bone formation, at the onset of secondary bone formation, and during coupled remodeling, respectively. Control animals received saline injections. mBMPR1A-mFC treatment initiated on day 1 delayed cartilage maturation in the callus and resulted in large regions of fibrous tissue. Treatment initiated on day 1 also increased the amount of mineralized tissue and up-regulated many bone-associated genes (p = 0.002) but retarded periosteal bony bridging and impaired strength and toughness at day 35 (p < 0.035). Delaying the onset of treatment to day 14 or 21 partially mitigated these effects and produced evidence of accelerated coupled remodeling. These results indicate that inhibition of the BMPR1A-mediated signaling has negative effects on secondary fracture healing that are differentially manifested at different stages of healing and within different cell populations. These effects are most pronounced during the endochondral period and appear to be mediated by selective inhibition of BMPRIA signaling within the periosteum. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:2096-2105, 2016.

Tendon healing is characterized by the formation of fibrovascular scar tissue, as tendon has very little intrinsic regenerative capacity. This creates a substantial clinical challenge in the setting of large, chronic tears seen clinically. Interest in regenerative healing seen in amphibians and certain strains of mice has arisen in response to the biological behavior of tendon tissue. Bone is also a model of tissue regeneration as healing bone will achieve the mechanical and histologic characteristics of the original tissue. The ultimate goal of the study of genes and mechanisms that contribute to true tissue regeneration is to ultimately attempt to manipulate the expression of those genes and activate these mechanisms in the setting of tendon injury and repair. Clearly, further research is needed to bring this to the forefront, however, study of scarless healing has potential to have meaningful application to tendon healing.

A targeted proteomic analysis of murine serum over a 35-day course of fracture healing was carried out to determine if serum proteomic changes could be used to monitor the biological progression of fracture healing. Transverse, closed femoral fractures where generated and stabilized with intramedullary fixation. A single stranded DNA aptamer-based multiplexed proteomic approach was used to assay 1,310 proteins. The transcriptomic profiles for genes matching the 1,310 proteins were obtained by microarray analysis of callus mRNA. Of the 1,310 proteins analyzed, 850 proteins showed significant differences among the time points (p-value

Radiographic Union Score for Tibia (RUST) and modified RUST (mRUST) are radiographic tools for quantitatively evaluating fracture healing using a cortical scoring system. This tool has high intra-class correlation coefficients (ICCs); however, little evidence has evaluated the scores against the physical properties of bone healing. Closed, stabilized fractures were made in the femora of C3H/HeJ male mice (8-12 week-old) of two dietary groups: a control and a phosphate restricted diet group. Micro-computed tomography (μCT) and torsion testing were carried out at post-operative days (POD) 14, 21, 35, and 42 (n=10-16) per group time-point. Anteroposterior and lateral radiographic views were constructed from the μCT scans and scored by five raters. The raters also indicated if the fracture were healed. ICCs were 0.71 (mRUST) and 0.63 (RUST). Both RUST scores were positively correlated with callus bone mineral density (BMD) (r = 0.85 and 0.80, p 0.318). Clinical relevance of this study is both RUST scores showed high correlation to physical properties of healing and generally distinguished healed vs. non-healed fractures. This article is protected by copyright. All rights reserved.

The relationship between BMP2 expression and the recruitment of skeletogenic stem cells was assessed following bone marrow reaming. BMP2 expression was examined using transgenic mice in which β-galactosidase had been inserted into the coding region of BMP2. Stem cell mobilization was analyzed by FACS analysis using CD73, a marker associated with bone marrow stromal stem cells. BMP2 expression was induced in endosteal lining cells, cortical osteocytes and periosteal cells in both the reamed and in contralateral bones. BMP2 mRNA expression in the reamed bone showed an early peak within the first 24 h of reaming followed by a later peak at 7 days, while contralateral bones only showed the 7 days peak of expression. FACS analysis sorting on CD73 positive cells showed a 50% increase of these cells at 3 and 14 days in the marrow of the injured bone and a single peak at 14 days of the marrow cell population of the contralateral bone. A ∼20% increase of CD73 positive cells was seen in the peripheral blood 2 days after reaming. These data showed that traumatic bone injury caused a systemic induction of BMP2 expression and that this increase is correlated with the mobilization of CD73 positive cells.

Assessment of the early stages of fracture healing via X-rays and computed tomography is limited by the low radio-opacity of cartilage. We validated a method of contrast-enhanced computed tomography (CECT) for non-destructive identification of cartilage within a healing fracture callus. Closed, stabilized fractures in femora of C57BL/6 mice were harvested on post-operative day 9.5 and imaged ex vivo with micro-computed tomography (µCT) before and after incubation in a cationic contrast agent that preferentially accumulates in cartilage due to the high concentration of sulfated glycosaminoglycans in the tissue. Co-registration of the pre- and post-incubation images, followed by image subtraction, enabled two- and three-dimensional delineation of mineralized tissue, soft callus, and cartilage. The areas of cartilage and callus identified with CECT were compared to those identified with the gold-standard method of histomorphometry. No difference was found between the areas of cartilage measured by the two methods (p = 0.999). Callus area measured by CECT was smaller than, but strongly predictive of (R(2) = 0.80, p < 0.001), the corresponding histomorphometric measurements. CECT also enabled qualitative identification of mineralized cartilage. These findings indicate that the CECT method provides accurate, quantitative, and non-destructive visualization of the shape and composition of the fracture callus, even during the early stages of repair when little mineralized tissue is present. The non-destructive nature of this method would allow subsequent analyses, such as mechanical testing, to be performed on the callus, thus enabling higher-throughput, comprehensive investigations of bone healing.

The purpose of this study was to establish a reliable model of metaphyseal bone healing and to use this model to investigate the effect of recombinant human osteogenic protein 1 (rhOP-1; BMP-7) and parathyroid hormone fragment (PTH 1-34) on healing. A wedge-shaped osteotomy was created in the distal tibia of 16-week-old female New Zealand White rabbits (n = 20) and was bridged with a custom-made external fixator. Five experimental groups of four animals each were investigated. In groups 1-4 the osteotomy gap was filled with tricalcium phosphate (TCP), and the gap was left unfilled in group 5 ("normal healing"). In group 1, 200 microg OP-1 was mixed in with the TCP. Groups 2 and 3 received daily subcutaneous injections of 10 and 40 microg/kg PTH, respectively, beginning on postoperative day 1. Radiographs were taken weekly. Following sacrifice on postoperative day 28, peripheral quantitative computed tomography (pQCT), histology, and mechanical testing (axial compression and torsion) were performed. Only one animal failed to complete the full 4-week time course, and no infections were encountered. Bone healing occurred in all animals. OP-1 stimulated bone formation locally, while the lower dose of PTH enhanced bone formation systemically (p < 0.05). Tibiae treated with OP-1 exhibited higher torsional strength (p = 0.04) than those in the normal healing group. These results indicate that a reliable and reproducible surgical model of metaphyseal healing has been established. In addition, differences in systemic versus local effects of PTH and OP-1 in accelerating metaphyseal fracture healing were found.

A new system for the local delivery of chemotherapy to malignant solid tumors has been developed based on calcium phosphate (CaP) nanoparticles. The adsorption of the anti-neoplastic drug cis-diamminedichloroplatinum (cisplatin) was characterized on three types of apatitic CaP (poorly and well crystallized hydroxyapatite, and carbonated apatite). Adsorption isotherms obtained in chloride-free phosphate solutions at pH = 7.4 (24 and 37 degrees C) indicate that cisplatin adsorption increases with temperature and increases with decreasing crystallinity. Release studies in phosphate buffer saline (containing the chloride ion essential for release) showed that while the cumulative amount of released drug was the same for all apatites at 20 days (approximately 70% of the total bound), the least crystalline material released the drug more slowly. The drug release rate increased slightly with temperature. Cytotoxicity testing was conducted in a K8 clonal murine osteosarcoma cell line to verify that drug activity was retained after adsorption onto the apatite crystals. K8 cells were plated onto dried films of the apatite/cisplatin conjugates and after 24 h, viability was measured with tritiated uridine. The apatite/cisplatin formulations exhibited cytotoxic effects with a dose dependent diminishment of cell viability.

Non-steroidal anti-inflammatory drugs (NSAIDs) specifically inhibit cyclooxygenase (COX) activity and are widely used as anti-arthritics, post-surgical analgesics, and for the relief of acute musculoskeletal pain. Recent studies suggest that non-specific NSAIDs, which inhibit both COX-1 and COX-2 isoforms, delay bone healing. The objectives of this study were 2-fold; first, to measure the relative changes in the normal expression of COX-1 and COX-2 mRNAs over a 42 day period of fracture healing and second, to compare the effects of a commonly used non-specific NSAID, ketorolac, with a COX-2 specific NSAID, Parecoxib (a pro-drug of valdecoxib), on this process. Simple, closed, transverse fractures were generated in femora of male Sprague-Dawley rats weighing approximately 450 g each. Total RNA was prepared from the calluses obtained prior to fracture and at 1, 3, 5, 7, 10, 14, 21, 35 and 42 days post-fracture and levels of COX-1 and COX-2 mRNA were measured using real time PCR. While the relative levels of COX-1 mRNA remained constant over a 21-day period, COX-2 mRNA levels showed peak expression during the first 14 days of healing and returned to basal levels by day 21. Mechanical properties of the calluses were then assessed at 21 and 35 days post-fracture in untreated animals and animals treated with either ketorolac or high or low dose parecoxib. At both 21 and 35 days after fracture, calluses in the group treated with the ketorolac showed a significant reduction in mechanical strength and stiffness when compared with controls (p

Distraction osteogenesis has proven to be of great value for the treatment of a variety of musculoskeletal problems. Little is still known, however, about the phenotypic changes in the cells participating in the bone formation process, induced by the procedure. Recent findings of the expression of a contractile muscle actin isoform, α-smooth muscle actin (SMA), in musculoskeletal connective tissue cells prompted this immunohistochemical study of the expression of SMA in cells participating in distraction osteogenesis in a rat model. The tissues within and adjacent to the distraction site could be distinguished histologically on the basis of cell morphology, density, and extracellular matrix make-up. The percentage of SMA-containing cells within each tissue zone was graded from 0 to 4. The majority of the cells in each of the zones stained positive for SMA within five days of the distraction period. The SMA-containing cells included those with elongated morphology in the center of the distraction site and the active osteoblasts on the surfaces of the newly forming bone. These finding warrant further investigation of the role of this contractile actin isoform in distraction osteogenesis and investigation of the effects of modulation of this actin isoform on the procedure.

Bone regeneration during fracture healing has been demonstrated repeatedly, yet the regeneration of articular cartilage and joints has not yet been achieved. It has been recognized however that the mechanical environment during fracture healing can be correlated to the contributions of either the endochondral or intramembranous processes of bone formation, and to resultant tissue architecture. Using this information, the goal of this study was to test the hypothesis that induced motion can directly regulate osteogenic and chondrogenic tissue formation in a rat mid-femoral bone defect and thereby influence the anatomical result. Sixteen male Sprague Dawley rats (400 +/- 20 g) underwent production of a mid-diaphyseal, non-critical sized 3.0 mm segmental femoral defect with rigid external fixation using a custom designed four pin fixator. One group of eight animals represented the controls and underwent surgery and constant rigid fixation. In the treatment group the custom external fixator was used to introduce daily interfragmentary bending strain in the eight treatment animals (12 degree angular excursion), with a hypothetical symmetrical bending load centered within the gap. The eight animals in the treatment group received motion at 1.0 Hz, for 10 min a day, with a 3 days on, one day off loading protocol for the first two weeks, and 2 days on, one day off for the remaining three weeks. Data collection included histological and immunohistological identification of tissue types, and mean collagen fiber angles and angular conformity between individual fibers in superficial, intermediate, and deep zones within the cartilage. These parameters were compared between the treatment group, rat knee articular cartilage, and the control group as a structural outcome assessment. After 35 days the control animals demonstrated varying degrees of osseous union of the defect with some animals showing partial union. In every individual within the mechanical treatment group the defect completely failed to unite. Bony arcades developed in the experimental group, capping the termini of the bone segments on both sides of the defect in four out of six animals completing the study. These new structures were typically covered with cartilage, as identified by specific histological staining for Type II collagen and proteoglycans. The distribution of collagen within analogous superficial, intermediate, and deep zones of the newly formed cartilage tissue demonstrated preferred fiber angles consistent with those seen in articular cartilage. Although not resulting in complete joint development, these neoarthroses show that the induced motion selectively controlled the formation of cartilage and bone during fracture repair, and that it can be specifically directed. They further demonstrate that the spatial organization of molecular components within the newly formed tissue, at both microanatomical and gross levels, are influenced by their local mechanical environment, confirming previous theoretical models.

The structural integrity of microfilaments has been shown to be necessary for the signal transduction of mechanical stimuli within osteoblasts. Qualitative and quantitative changes within the cytoskeleton of osteoblasts may therefore be crucial components of the signal transduction processes of these cells in response to mechanical stimulation. Avian osteoblasts were strained with a device that deforms a flexible, cell-laden membrane at a defined frequency and intensity in a uniform biaxial manner. We examined the effects of mechanical strain on the accumulation of protein and the expression of the major cytoskeletal elements and specific integrin-binding (arginine-glycine-aspartic acid) proteins of these cells. Mechanical strain increased the level of total extracellular matrix-accumulated fibronectin by approximately 150% and decreased that of osteopontin by approximately 60% but had no quantifiable effect on the accumulation of beta1 integrin subunit or collagen type I. An examination of the major elements of the cytoskeleton demonstrated that neither the level of actin nor that of the intermediate filament protein vimentin changed; however, the amount of tubulin decreased by approximately 75% and the amount of vinculin, a major protein of focal adhesion complexes, increased by approximately 250%. An analysis of protein synthesis by two-dimensional gel electrophoresis of [35S]methionine-labeled cytoskeletal proteins demonstrated that the changes in the accumulation of vinculin and tubulin resulted from their altered synthesis. Messenger RNA analysis confirmed that the changes in accumulation and protein synthesis observed for vinculin, fibronectin, and osteopontin were controlled at a pretranslational level. Immunofluorescent microscopy demonstrated that mechanical strain led to increased formation and thickening of actin stress fibers, with a commensurate dissociation in microtubules and a clear increase in levels of vinculin at the peripheral edges of the cells. In conclusion, the elevated rate of synthesis and the increased accumulation of vinculin and fibronectin, as well as the increase in the number and size of stress fibers and focal adhesion complexes, suggest that mechanical strain leads to a coordinated change both in the cytoskeleton and in extracellular matrix proteins that will facilitate tighter adhesion of an osteoblast to its extracellular matrix.