Cells transiently expressing periostin are required for intramedullary intramembranous bone regeneration.

Intramembranous bone regeneration plays an important role in fixation of intramedullary implants used in joint replacement and dental implants used in tooth replacement. Despite widespread recognition of the importance of intramembranous bone regeneration in these clinical procedures, the underlying mechanisms have not been well explored. A previous study that examined transcriptomic profiles of regenerating bone from the marrow space showed that increased periostin gene expression preceded increases in several osteogenic genes. We therefore sought to determine the role of cells transiently expressing periostin in intramedullary intramembranous bone regeneration. We used a genetic mouse model that allows tamoxifen-inducible fluorescent labeling of periostin expressing cells. These mice underwent ablation of the bone marrow cavity through surgical disruption, a well-established intramembranous bone regeneration model. We found that in intact bones, fluorescently labeled cells were largely restricted to the periosteal surface of cortical bone and were absent in bone marrow. However, following surgical disruption of the bone marrow cavity, cells transiently expressing periostin were found within the regenerating tissue of the bone marrow compartment even though the cortical bone remained intact. The source of these cells is likely heterogenous, including cells occupying the periosteal surface as well as pericytes and endothelial cells within the marrow cavity. We also found that diphtheria toxin-mediated depletion of cells transiently expressing periostin at the time of surgery impaired intramembranous bone regeneration in mice. These data suggest a critical role of periostin expressing cells in intramedullary intramembranous bone regeneration and may lead to novel therapeutic interventions to accelerate or enhance implant fixation.

SHP-1 Protein Tyrosine Phosphatase Affects Early Postnatal Bone Development in Mice.

The Src homology region 2 domain-containing phosphatase-1 (SHP-1) is an intracellular tyrosine phosphatase that plays a negative regulatory role in immune cell signaling. Absent or diminished SHP-1 catalytic activity results in reduced bone mass with enhanced bone resorption. Here, we sought to investigate if Shp1 overexpression leads to increased bone mass and improved mechanical properties. Male and female wildtype (WT) and SHP1-transgenic (Tg) mice at 28, 56, and 84 days of age were compared. We applied microcomputed tomography to assess femoral cortical bone geometry and trabecular architecture and 3-point mechanical bending to assess mid-diaphyseal structural and estimated material properties. Serum OPG, RANKL, P1NP, and CTX-1 concentrations were measured by enzyme-linked immunoassay. The majority of transgene effects were restricted to the 28-day-old mice. Trabecular bone volume per total volume, trabecular number, and connectivity density were greater in 28-day-old female SHP1-Tg mice when compared to WTs. SHP1-Tg female mice showed increased total and medullary areas, with no difference in cortical area and thickness. Cortical tissue mineral density was strongly genotype-dependent. Failure load, yield load, ultimate stress, and yield stress were all lower in 28-day-old SHP1-Tg females. In 28-day-old SHP1-Tg females, circulating levels of OPG and P1NP were higher and RANKL levels were lower than WT controls. Our study demonstrates a role for SHP-1 in early postnatal bone development; SHP-1 overexpression negatively impacted whole bone strength and material properties in females.

Contrast-enhanced micro-computed tomography of compartment and time-dependent changes in femoral cartilage and subchondral plate in a murine model of osteoarthritis.

A lack of understanding of the mechanisms underlying osteoarthritis (OA) progression limits the development of effective long-term treatments. Quantitatively tracking spatiotemporal patterns of cartilage and bone degeneration is critical for assessment of more appropriately targeted OA therapies. In this study, we use contrast-enhanced micro-computed tomography (µCT) to establish a timeline of subchondral plate (SCP) and cartilage changes in the murine femur after destabilization of the medial meniscus (DMM). We performed DMM or sham surgery in 10-12-week-old male C57Bl/6J mice. Femora were imaged using µCT after 0, 2, 4, or 8 weeks. Cartilage-optimized scans were performed after immersion in contrast agent CA4+. Bone mineral density distribution (BMDD), cartilage attenuation, SCP, and cartilage thickness and volume were measured, including lateral and medial femoral condyle and patellar groove compartments. As early as 2 weeks post-DMM, cartilage thickness significantly increased and cartilage attenuation, SCP volume, and BMDD mean significantly decreased. Trends in cartilage and SCP metrics within each joint compartment reflected those seen in global measurements, and both BMDD and SCP thickness were consistently greater in the lateral and medial condyles than the patellar groove. Sham surgery also resulted in significant changes to SCP and cartilage metrics, highlighting a potential limitation of using surgical models to study tissue morphology or composition changes during OA progression. Contrast-enhanced µCT analysis is an effective tool to monitor changes in morphology and composition of cartilage, and when combined with bone-optimized µCT, can be used to assess the progression of degenerative changes after joint injury.

Colon epithelial cell-specific Bmal1 deletion impairs bone formation in mice.

The circadian clock system regulates multiple metabolic processes, including bone metabolism. Previous studies have demonstrated that both central and peripheral circadian signaling regulate skeletal growth and homeostasis in mice. Disruption in central circadian rhythms has been associated with a decline in bone mineral density in humans and the global and osteoblast-specific disruption of clock genes in bone tissue leads to lower bone mass in mice. Gut physiology is highly sensitive to circadian disruption. Since the gut is also known to affect bone remodeling, we sought to test the hypothesis that circadian signaling disruption in colon epithelial cells affects bone. We therefore assessed structural, functional, and cellular properties of bone in 8 week old Ts4-Cre and Ts4-Cre;Bmal1fl/fl (cBmalKO) mice, where the clock gene Bmal1 is deleted in colon epithelial cells. Axial and appendicular trabecular bone volume was significantly lower in cBmalKO compared to Ts4-Cre 8-week old mice in a sex-dependent fashion, with male but not female mice showing the phenotype. Similarly, the whole bone mechanical properties were deteriorated in cBmalKO male mice. The tissue level mechanisms involved suppressed bone formation with normal resorption, as evidenced by serum markers and dynamic histomorphometry. Our studies demonstrate that colon epithelial cell-specific deletion of Bmal1 leads to failure to acquire trabecular and cortical bone in male mice.

Activation of canonical Wnt signaling accelerates intramembranous bone regeneration in male mice.

Canonical Wnt signaling plays an important role in skeletal development, homeostasis, and both endochondral and intramembranous repair. While studies have demonstrated that the inhibition of Wnt signaling impairs intramembranous bone regeneration, how its activation affects intramembranous bone regeneration has been underexplored. Therefore, we sought to determine the effects of activation of canonical Wnt signaling on intramembranous bone regeneration by using the well-established marrow ablation model. We hypothesized that mice with a mutation in the Wnt ligand coreceptor gene Lrp5 would have accelerated intramembranous bone regeneration. Male and female wild-type and Lrp5-mutant mice underwent unilateral femoral bone marrow ablation surgery in the right femur at 4 weeks of age. Both the left intact and right operated femurs were assessed at Days 3, 5, 7, 10, and 14. The intact femur of Lrp5 mutant mice of both sexes had higher bone mass than wild-type littermates, although to a greater degree in males than females. Overall, the regenerated bone volume in Lrp5 mutant male mice was 1.8-fold higher than that of littermate controls, whereas no changes were observed between female Lrp5 mutant and littermate control mice. In addition, the rate of intramembranous bone regeneration (from Day 3 to Day 7) was higher in Lrp5 mutant male mice compared to their same-sex littermate controls with no difference in the females. Thus, activation of canonical Wnt signaling increases bone mass in intact bones of both sexes, but accelerates intramembranous bone regeneration following an injury challenge only in male mice.

The risk of revision following total hip arthroplasty in patients with inflammatory bowel disease, a registry based study.

Inflammatory bowel disease (IBD) is characterized by chronic inflammation of the intestinal tract and is associated with decreased bone mineral density. IBD patients are at higher risk of osteopenia, osteoporosis and fracture compared to non-IBD patients. The impact of IBD on the performance of orthopedic implants has not been well studied. We hypothesized that a history of IBD at the time of primary total hip arthroplasty (THA) would increase the risk of subsequent failure as assessed by revision surgery. A retrospective implant survival analysis was completed using the Swedish Hip Arthroplasty Registry and the Sweden National Patient Register. A total of 150,073 patients undergoing THA for osteoarthritis within an 18-year period were included in the study. THA patients with (n = 2,604) and without (n = 147,469) a history of IBD at the time of THA were compared with primary revision as the main endpoint and adjusted using sex, age category and comorbidity (Elixhauser scores) as covariates. We found that patients with a history of IBD had a relatively higher risk of revision surgery for septic causes while the non-IBD patients had a relatively higher risk of revision for aseptic causes (p = 0.004). Our findings suggest there may be an association between gut health and THA performance.

How faithfully does intramembranous bone regeneration recapitulate embryonic skeletal development?

Postnatal intramembranous bone regeneration plays an important role during a wide variety of musculoskeletal regeneration processes such as fracture healing, joint replacement and dental implant surgery, distraction osteogenesis, stress fracture healing, and repair of skeletal defects caused by trauma or resection of tumors. The molecular basis of intramembranous bone regeneration has been interrogated using rodent models of most of these conditions. These studies reveal that signaling pathways such as Wnt, TGFß/BMP, FGF, VEGF, and Notch are invoked, reminiscent of embryonic development of membranous bone. Discoveries of several skeletal stem cell/progenitor populations using mouse genetic models also reveal the potential sources of postnatal intramembranous bone regeneration. The purpose of this review is to compare the underlying molecular signals and progenitor cells that characterize embryonic development of membranous bone and postnatal intramembranous bone regeneration.

CHIP regulates skeletal development and postnatal bone growth.

C terminus of Hsc70-interacting protein (CHIP) is a chaperone-dependent and U-box containing E3 ubiquitin ligase. In previous studies, we found that CHIP regulates the stability of multiple tumor necrosis factor receptor-associated factor proteins in bone cells. In Chip global knockout (KO) mice, nuclear factor-κB signaling is activated, osteoclast formation is increased, osteoblast differentiation is inhibited, and bone mass is decreased in postnatal Chip KO mice. To determine the role of Chip in different cell types at different developmental stages, we created Chipflox/flox mice. We then generated Chip conditional KO mice ChipCMV and ChipOsxER and demonstrated defects in skeletal development and postnatal bone growth in Chip conditional KO mice. Our findings indicate that Chip conditional KO mice could serve as a critical reagent for further investigations of functions of CHIP in bone cells and in other cell types.

Chemically modified RNA activated matrices enhance bone regeneration.

There exists a dire need for improved therapeutics to achieve predictable bone regeneration. Gene therapy using non-viral vectors that are safe and efficient at transfecting target cells is a promising approach to overcoming the drawbacks of protein delivery of growth factors. Here, we investigated the transfection efficiency, cytotoxicity, osteogenic potential and in vivo bone regenerative capacity of chemically modified ribonucleic acid (cmRNA) (encoding BMP-2) complexed with polyethylenimine (PEI) and made comparisons with PEI complexed with conventional plasmid DNA (encoding BMP-2). The polyplexes were fabricated at an amine (N) to phosphate (P) ratio of 10 and characterized for transfection efficiency using human bone marrow stromal cells (BMSCs). The osteogenic potential of BMSCs treated with these polyplexes was validated by determining the expression of bone-specific genes, osteocalcin and alkaline phosphatase as well as through the detection of bone matrix deposition. Using a calvarial bone defect model in rats, it was shown that PEI-cmRNA (encoding BMP-2)-activated matrices promoted significantly enhanced bone regeneration compared to PEI-plasmid DNA (BMP-2)-activated matrices. Our proof of concept study suggests that scaffolds loaded with non-viral vectors harboring cmRNA encoding osteogenic proteins may be a powerful tool for stimulating bone regeneration with significant potential for clinical translation.

Assessment of glenoid chondral healing: comparison of microfracture to autologous matrix-induced chondrogenesis in a novel rabbit shoulder model.

BACKGROUND: Management of glenohumeral arthrosis in young patients is a considerable challenge, with a growing need for non-arthroplasty alternatives. The objectives of this study were to develop an animal model to study glenoid cartilage repair and to compare surgical repair strategies to promote glenoid chondral healing. METHODS: Forty-five rabbits underwent unilateral removal of the entire glenoid articular surface and were divided into 3 groups--untreated defect (UD), microfracture (MFx), and MFx plus type I/III collagen scaffold (autologous matrix-induced chondrogenesis [AMIC])--for the evaluation of healing at 8 weeks (12 rabbits) and 32 weeks (33 rabbits) after injury. Contralateral shoulders served as unoperated controls. Tissue assessments included 11.7-T magnetic resonance imaging (long-term healing group only), equilibrium partitioning of an ionic contrast agent via micro-computed tomography (EPIC-µCT), and histologic investigation (grades on International Cartilage Repair Society II scoring system). RESULTS: At 8 weeks, x-ray attenuation, thickness, and volume did not differ by treatment group. At 32 weeks, the T2 index (ratio of T2 values of healing to intact glenoids) was significantly lower for the MFx group relative to the AMIC group (P = .01) whereas the T1ρ index was significantly lower for AMIC relative to MFx (P = .01). The micro-computed tomography-derived repair tissue volume was significantly higher for MFx than for UD. Histologic investigation generally suggested inferior healing in the AMIC and UD groups relative to the MFx group, which exhibited improvements in both integration of repair tissue with subchondral bone and tidemark formation over time. DISCUSSION: Improvements conferred by AMIC were limited to magnetic resonance imaging outcomes, whereas MFx appeared to promote increased fibrous tissue deposition via micro-computed tomography and more hyaline-like repair histologically. The findings from this novel model suggest that MFx promotes biologic resurfacing of full-thickness glenoid articular injury.

A Pilot Study Evaluating Combinatorial and Simultaneous Delivery of Polyethylenimine-Plasmid DNA Complexes Encoding for VEGF and PDGF for Bone Regeneration in Calvarial Bone Defects.

Gene therapy is a promising strategy to deliver growth factors of interest locally in a sustained fashion and has the potential to overcome barriers to using recombinant protein therapy such as sustainability and cost. Recent studies demonstrate the safety and efficacy of non-viral delivery of plasmid DNA (pDNA) encoding a single growth factor to enhance bone healing. This pilot study is aimed at testing a non-viral gene delivery system that can deliver two different plasmids encoding two different growth factors. Polyethylenimine (PEI), a cationic polymer, was utilized as a gene delivery vector and collagen scaffold was used as a carrier to deliver the PEI-pDNA complexes encoding platelet derived growth factor B (PDGF-B) and/or vascular endothelial growth factor (VEGF). Calvarial defects in rats were implanted with scaffolds containing PEI-pPDGF-B complexes, PEI-pVEGF complexes or containing both PEIpPDGF- B and PEI-pVEGF complexes in a 1:1 ratio of plasmids. The results indicated that bone regeneration as measured using micro-CT and histological assessments was inferior in groups treated with PEI-(pPDGF-B + pVEGF) complexes, compared to defects treated with PEI-pPDGF-B complexes. This pilot study that explores the feasibility and efficacy of combinatorial non-viral gene delivery system for bone regeneration appears to provide a rationale for investigation of sequential delivery of growth factors at specific time points during the healing phases and this will be explored further in future studies.

Are there biological markers for wear or corrosion? A systematic review.

BACKGROUND: Identification of biomarkers associated with wear and tribocorrosion in joint arthroplasty would be helpful to enhance early detection of aseptic loosening and/or osteolysis and to improve understanding of disease progression. There have been several new reports since the last systematic review (which covered research through mid-2008) justifying a new assessment. QUESTIONS/PURPOSES: We sought to determine which biomarkers have the most promise for early diagnosis and monitoring of aseptic loosening and/or osteolysis related to wear or corrosion in total joint arthroplasty. METHODS: We performed a systematic review using MEDLINE and EMBASE databases, covering the period through December 2013, and identified 1050 articles. We restricted the definition of biomarker to biomolecules and imaging parameters useful for diagnosis and monitoring of disease progression, only including articles in English. We chose 65 articles for full review, including 44 from the original search and 21 from subsequent hand searches. We used the 22 articles in which patients with total joint arthroplasty who had aseptic loosening and/or periimplant osteolysis unrelated to sepsis had been compared with patients with total joint arthroplasty with stable implants. There were 90 comparisons of these two patient populations involving 35 different biomarkers. RESULTS: Diagnostic accuracy was assessed in nine of the 90 comparisons with the highest accuracy found for tartrate-resistant acid phosphatase 5b (0.96), although a separate comparison for this biomarker found a lower accuracy (0.76). Accuracy of > 0.80 was also found for crosslinked n-telopeptide of type I collagen, osteoprotegerin, and deoxypyridinoline. The most studied markers, tumor necrosis factor-α and interleukin-1ß, were found to differ in the affected and control groups in < 30% of the comparisons. Thirty of the 35 biomarkers were studied in four or fewer separate comparisons with nearly half of the biomarkers (17) studied in only one comparison. Many of the comparisons were not able to eliminate a number of confounding variables, and there was only one prospective study. CONCLUSIONS: Currently, there are no validated biomarkers for early diagnosis and monitoring of the biological sequelae of wear or tribocorrosion, although there are some promising leads, including markers of bone turnover.

The enhancement of bone regeneration by gene activated matrix encoding for platelet derived growth factor.

Gene therapy using non-viral vectors that are safe and efficient in transfecting target cells is an effective approach to overcome the shortcomings of protein delivery of growth factors. The objective of this study was to develop and test a non-viral gene delivery system for bone regeneration utilizing a collagen scaffold to deliver polyethylenimine (PEI)-plasmid DNA (pDNA) [encoding platelet derived growth factor-B (PDGF-B)] complexes. The PEI-pPDGF-B complexes were fabricated at amine (N) to phosphate (P) ratio of 10 and characterized for size, surface charge, and in vitro cytotoxicity and transfection efficacy in human bone marrow stromal cells (BMSCs). The influence of the complex-loaded collagen scaffold on cellular attachment and recruitment was evaluated in vitro using microscopy techniques. The in vivo regenerative capacity of the gene delivery system was assessed in 5 mm diameter critical-sized calvarial defects in Fisher 344 rats. The complexes were ~100 nm in size with a positive surface charge. Complexes prepared at an N/P ratio of 10 displayed low cytotoxicity as assessed by a cell viability assay. Confocal microscopy revealed significant proliferation of BMSCs on complex-loaded collagen scaffolds compared to empty scaffolds. In vivo studies showed significantly higher new bone volume/total volume (BV/TV) % in calvarial defects treated with the complex-activated scaffolds following 4 weeks of implantation (14- and 44-fold higher) when compared to empty defects or empty scaffolds, respectively. Together, these findings suggest that non-viral PDGF-B gene-activated scaffolds are effective for bone regeneration and are an attractive gene delivery system with significant potential for clinical translation.

Temporal gene expression profiling during rat femoral marrow ablation-induced intramembranous bone regeneration.

Enhanced understanding of differential gene expression and biological pathways associated with distinct phases of intramembranous bone regeneration following femoral marrow ablation surgery will improve future advancements regarding osseointegration of joint replacement implants, biomaterials design, and bone tissue engineering. A rat femoral marrow ablation model was performed and genome-wide microarray data were obtained from samples at 1, 3, 5, 7, 10, 14, 28, and 56 days post-ablation, with intact bones serving as controls at Day 0. Bayesian model-based clustering produced eight distinct groups amongst 9,062 significant gene probe sets based on similar temporal expression profiles, which were further categorized into three major temporal classes of increased, variable, and decreased expression. Osteoblastic- and osteoclastic-associated genes were found to be significantly expressed within the increased expression groups. Chondrogenesis was not detected histologically. Adipogenic marker genes were found within variable/decreased expression groups, emphasizing that adipogenesis was inhibited during osteogenesis. Differential biological processes and pathways associated with each major temporal group were identified, and significantly expressed genes involved were visually represented by heat maps. It was determined that the increased expression group exclusively contains genes involved in pathways for matrix metalloproteinases (MMPs), Wnt signaling, TGF-ß signaling, and inflammatory pathways. Only the variable expression group contains genes associated with glycolysis and gluconeogenesis, the notch signaling pathway, natural killer cell mediated cytotoxicity, and the B cell receptor signaling pathway. The decreased group exclusively consists of genes involved in heme biosynthesis, the p53 signaling pathway, and the hematopoietic cell lineage. Significant biological pathways and transcription factors expressed at each time point post-ablation were also identified. These data present the first temporal gene expression profiling analysis of the rat genome during intramembranous bone regeneration induced by femoral marrow ablation.

Co-Cr-Mo alloy particles induce tumor necrosis factor alpha production in MLO-Y4 osteocytes: a role for osteocytes in particle-induced inflammation.

Wear debris-induced osteolysis is purportedly the limiting problem affecting the long term results of joint arthroplasty. Pathogenic effects of wear debris in peri-implant cells such as macrophages, osteoblasts and osteoclasts have been well studied. In contrast, the effects of wear debris on osteocytes, which make up over 90% of all bone cells, remain unknown. We hypothesized that metal implant debris can induce the pro-inflammatory response in osteocytes. This study demonstrated the effects of cobalt-chromium-molybdenum alloy (Co-Cr-Mo) particles on a well-characterized MLO-Y4 osteocyte cell line. Co-Cr-Mo alloy particle treatment significantly (p<0.05) up-regulated tumor necrosis factor alpha (TNFalpha) gene expression after 3 and 6 h and TNFalpha protein production after 24 h, but down-regulated interleukin-6 (IL-6) gene expression after 6 h. Co-Cr-Mo alloy particle treatment also induced osteocyte apoptosis after 24 h. This apoptotic effect was partially (40%) dependent on TNFalpha. Therefore, our results suggest that osteocytes play a role in particle-induced inflammation and bone resorption following total joint arthroplasty by inducing pro-inflammatory cytokines and inducing osteocyte apoptosis.

Porous implants as drug delivery vehicles to augment host tissue integration.

The common premise of synthetic implants in the restoration of diseased tissues and organs is to use inert and solid materials. Here, a porous titanium implant was fabricated for the delivery of microencapsulated bioactive cues. Control-released transforming growth factor-beta1 (TGF-beta1) promoted the proliferation and migration of human mesenchymal stem cells into porous implants in vitro. At 4 wk of implantation in the rabbit humerus, control-released TGF-beta1 from porous implants significantly increased bone-to-implant contact (BIC) by 96% and bone ingrowth by 50% over placebos. Control-released 100 ng TGF-beta1 induced equivalent BIC and bone ingrowth to adsorbed 1 microg TGF-beta1, suggesting that controlled release is effective at 10-fold less drug dose than adsorption. Histomorphometry, scanning electron microscopy, and microcomputed tomography showed that control-released TGF-beta1 enhanced bone ingrowth in the implant's pores and surface. These findings suggest that solid prostheses can be transformed into porous implants to serve as drug delivery carriers, from which control-released bioactive cues augment host tissue integration.

Autologous stem cell regeneration in craniosynostosis.

Craniosynostosis occurs in one of 2500 live human births and may manifest as craniofacial disfiguration, seizure, and blindness. Craniotomy is performed to reshape skull bones and resect synostosed cranial sutures. We demonstrate for the first time that autologous mesenchymal stem cells (MSCs) and controlled-released TGFbeta3 reduced surgical trauma to localized osteotomy and minimized osteogenesis in a rat craniosynostosis model. Approximately 0.5 mL tibial marrow content was aspirated to isolate mononucleated and adherent cells that were characterized as MSCs. Upon resecting the synostosed suture, autologous MSCs in collagen carriers with microencapsulated TGFbeta3 (1 ng/mL) generated cranial suture analogs characterized as bone-soft tissue-bone interface by quantitative histomorphometric and microCT analyses. Thus, surgical trauma in craniosynostosis can be minimized by a biologically viable implant. We speculate that proportionally larger amounts of human marrow aspirates participate in the healing of craniosynostosis defects in patients. The engineered soft tissue-bone interface may have implications in the repair of tendons, ligaments, periosteum and periodontal ligament.