Human Fracture Healing Research Articles

Activation of Wnt signaling in human fracture callus and nonunion tissues

The Wnt signaling pathway is a key molecular process during fracture repair. Although much of what we now know about the role of this pathway in bone is derived from in vitro and animal studies, the same cannot be said about humans. As such, we hypothesized that Wnt signaling will also be a key process in humans during physiological fracture healing as well as in the development of a nonunion (hypertrophic and oligotrophic). We further hypothesized that the expression of Wnt-signaling pathway genes/proteins would exhibit a differential expression pattern between physiological fracture callus and the pathological nonunion tissues. We tested these two hypotheses by examining the mRNA levels of key Wnt-signaling related genes: ligands (WNT4, WNT10a), receptors (FZD4, LRP5, LRP6), inhibitors (DKK1, SOST) and modulators (CTNNB1 and PORCN). RNA sequencing from calluses as well as from the two nonunion tissue types, revealed that all of these genes were expressed at about the same level in these three tissue types. Further, spatial expression experiments identified the cells responsible of producing these proteins. Robust expression was detected in osteoblasts for the majority of these genes except SOST which displayed low expression, but in contrast, was mostly detected in osteocytes. Many of these genes were also expressed by callus chondrocytes as well. Taken together, these results confirm that Wnt signaling is indeed active during both human physiological fracture healing as well as in pathological nonunions.

Close negative correlation of local and circulating Dickkopf-1 and Sclerostin levels during human fracture healing

Wnt signaling is critically involved in fracture healing. Existing data predominantly relies on rodent models. Here, we explored local and circulating Dickkopf-1 (DKK1) levels in patients with respect to fracture healing and explore its association to sclerostin (SOST). 69 patients after surgical stabilization of long bone fractures of which six patients had impaired fracture healing were included in this study. Life-style and patient related factors with a known effect on DKK1 and SOST were recorded. DKK1 and SOST concentrations were measured using enzyme-linked immunosorbent assay (ELISA) at the fracture site and in circulation. DKK1 and SOST showed a close inverse correlation. In fracture hematoma and immediately after trauma DKK1 levels were significantly reduced while SOST levels were significantly increased, compared to healthy control. Postoperatively, DKK1 peaked at week 2 and SOST at week 8, again demonstrating a close negative correlation. Age and smoking status affected the balance of DKK1 and SOST, while type 2 diabetes and sex did not demonstrate a significant influence. Early postoperative elevation of SOST without compensatory DKK1 decrease was associated with fracture non-union in younger patients (< 50a). The close inverse correlation and very rapid dynamics of DKK1 and SOST locally as well as systemically suggest their critical involvement during human fracture healing. Importantly, as immediate compensatory feedback mechanism are apparent, we provide evidence that dual-blockade of DKK1 and SOST could be critical to allow for therapeutic efficiency of Wnt targeted therapies for fracture healing.

Impact Of Connective Tissue Matrix Products and Critical Size Defects on Gut Microbiota and Fracture Healing in Mice

Abstract: &#x0D; Approximately 6.2 million people in the USA alone suffer from some form of fracture annually. Depending on the intensity of trauma and complexity of the fractures, some fractures will not heal without medical intervention. It is imperative to develop novel therapies that target fracture healing. Connective Tissue Matrix (CTM) Biomedical is a company that develops human placenta, amnion, chorion, and other umbilical derived allograft products which may provide a solution to the problem of impaired fracture healing. CTM products contain structural proteins, cytokines, and growth factors that may have regenerative and anti-inflammatory properties. Although these products are already used clinically for fracture and wound healing in humans, no pre-clinical studies exist verifying their efficacy or mechanism of action.&#x0D; Commensal microbiota is a collection of microorganisms such as bacteria, fungi, and viruses. Disruption of the gut microbiota, known as gut dysbiosis, can lead to a variety of disorders in other tissues. In fact, new studies indicate that alterations in the gut microbiota can affect bone health. This is likely due to impaired nutrient uptake and an increase in inflammation from bacterial byproducts that are not favorable to a healthy gut ecosystem. More recent studies indicate that the microbiota is also implicated in fracture healing.&#x0D; Our study aims to investigate if CTM products have a positive effect on fracture healing by affecting the gut microbiota composition and bacteriome. To test this, critical sized defects (CSD) were induced in mice and treated with various CTM implants or saline control. Fecal samples were taken on the day of surgery and weekly thereafter. Bacterial DNA was subsequently extracted from these samples and analyzed using PCR. We anticipate seeing changes in load and composition of gut bacteria following CSD surgery.

Activin A marks a novel progenitor cell population during fracture healing and reveals a therapeutic strategy.

Insufficient bone fracture repair represents a major clinical and societal burden and novel strategies are needed to address it. Our data reveal that the transforming growth factor-β superfamily member Activin A became very abundant during mouse and human bone fracture healing but was minimally detectable in intact bones. Single-cell RNA-sequencing revealed that the Activin A-encoding gene Inhba was highly expressed in a unique, highly proliferative progenitor cell (PPC) population with a myofibroblast character that quickly emerged after fracture and represented the center of a developmental trajectory bifurcation producing cartilage and bone cells within callus. Systemic administration of neutralizing Activin A antibody inhibited bone healing. In contrast, a single recombinant Activin A implantation at fracture site in young and aged mice boosted: PPC numbers; phosphorylated SMAD2 signaling levels; and bone repair and mechanical properties in endochondral and intramembranous healing models. Activin A directly stimulated myofibroblastic differentiation, chondrogenesis and osteogenesis in periosteal mesenchymal progenitor culture. Our data identify a distinct population of Activin A-expressing PPCs central to fracture healing and establish Activin A as a potential new therapeutic tool.

Computer-Based Mechanobiological Fracture Healing Model Predicts Non-Union of Surgically Treated Diaphyseal Femur Fractures.

As non-unions are still common, a predictive assessment of healing complications could enable immediate intervention before negative impacts for the patient occur. The aim of this pilot study was to predict consolidation with the help of a numerical simulation model. A total of 32 simulations of patients with closed diaphyseal femoral shaft fractures treated by intramedullary nailing (PFNA long, FRN, LFN, and DePuy Synthes) were performed by creating 3D volume models based on biplanar postoperative radiographs. An established fracture healing model, which describes the changes in tissue distribution at the fracture site, was used to predict the individual healing process based on the surgical treatment performed and full weight bearing. The assumed consolidation as well as the bridging dates were retrospectively correlated with the clinical and radiological healing processes. The simulation correctly predicted 23 uncomplicated healing fractures. Three patients showed healing potential according to the simulation, but clinically turned out to be non-unions. Four out of six non-unions were correctly detected as non-unions by the simulation, and two simulations were wrongfully diagnosed as non-unions. Further adjustments of the simulation algorithm for human fracture healing and a larger cohort are necessary. However, these first results show a promising approach towards an individualized prognosis of fracture healing based on biomechanical factors.

Vitamin D Status and Fracture Healing: A Systematic Review of 63 Studies

Category: Other Introduction/Purpose: There is little consensus on the effects of vitamin D on fracture healing. This review aims to clarify that effect in addition to determining trends in the classification of deficiency and supplementation. Methods: This systematic review searched Pubmed, Embase, and Web of Science for manuscripts related to vitamin D status/deficiency and fracture healing. Two independent reviewers screened articles, graded evidence quality, and extracted data. A total of 38 human studies, 11 in vivo studies and 14 systematic reviews were analyzed. Results: The median vitamin D deficiency prevalence reported amongst the human studies was 41.2% which is comparable to subset of patients with deficiency defined as &lt;20 ng/mL, 43.3%. Amongst the 9 human studies reporting vitamin D supplementation, the exact dosage varied greatly. No RCTs reported any radiographic evidence of increased fracture union with any form of supplementation. Several RCTs reported reduction in pain with smaller daily supplementation compared to placebo or single large boluses. Animal studies reported mixed results, with some showing no significant effects of vitamin D status on the biomechanical properties of bone, while others demonstrated a correlation between supplementation improved biomechanical stiffness. Furthermore, the metabolite 24,25D may be a mediator in Vitamin D's probable osteoprotective effects. Conclusion: While the role of vitamin D in fracture healing appears to be osteoprotective in animal models, the degree and significance of its effects on human fracture healing and prevention remains inconclusive. We recommend standardizing the definitions for vitamin D deficiency to be &lt;20 ng/mL for deficiency and &gt;30 ng/mL for sufficiency. Although there is no radiographic evidence, daily smaller doses of vitamin D supplementation seem to have a stronger impact on clinical fracture healing than large boluses.

Cross-species comparisons reveal resistance of human skeletal stem cells to inhibition by non-steroidal anti-inflammatory drugs.

Fracture healing is highly dependent on an early inflammatory response in which prostaglandin production by cyclo-oxygenases (COX) plays a crucial role. Current patient analgesia regimens favor opioids over Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) since the latter have been implicated in delayed fracture healing. While animal studies broadly support a deleterious role of NSAID treatment to bone-regenerative processes, data for human fracture healing remains contradictory. In this study, we prospectively isolated mouse and human skeletal stem cells (SSCs) from fractures and compared the effect of various NSAIDs on their function. We found that osteochondrogenic differentiation of COX2-expressing mouse SSCs was impaired by NSAID treatment. In contrast, human SSCs (hSSC) downregulated COX2 expression during differentiation and showed impaired osteogenic capacity if COX2 was lentivirally overexpressed. Accordingly, short- and long-term treatment of hSSCs with non-selective and selective COX2 inhibitors did not affect colony forming ability, chondrogenic, and osteogenic differentiation potential in vitro. When hSSCs were transplanted ectopically into NSG mice treated with Indomethacin, graft mineralization was unaltered compared to vehicle injected mice. Thus, our results might contribute to understanding species-specific differences in NSAID sensitivity during fracture healing and support emerging clinical data which conflicts with other earlier observations that NSAID administration for post-operative analgesia for treatment of bone fractures are unsafe for patients.

Electrical Stimulation of Acute Fractures: A Narrative Review of Stimulation Protocols and Device Specifications.

Orthopedic fractures have a significant impact on patients in the form of economic loss and functional impairment. Beyond the standard methods of reduction and fixation, one adjunct that has been explored since the late 1970s is electrical stimulation. Despite robust evidence for efficacy in the preclinical arena, human trials have mixed results, and this technology is not widely accepted. The purpose of this review is to examine the body of literature supporting electrical stimulation for the purpose of fracture healing in humans with an emphasis on device specifications and stimulation protocols and delineate a minimum reporting checklist for future studies of this type. We have isolated 12 studies that pertain to the administration of electrical stimulation for the purpose of augmenting fracture healing in humans. Of these, one was a direct current electrical stimulation study. Six studies utilized pulsed electromagnetic field therapy and five used capacitive coupling. When examining these studies, the device specifications were heterogenous and often incomplete in what they reported, which rendered studies unrepeatable. The stimulation protocols also varied greatly study to study. To demonstrate efficacy of electrical stimulation for fractures, the authors recommend isolating a fracture type that is prone to nonunion to maximize the electrical stimulation effect, a homogenous study population so as to not dilute the effect of electrical stimulation, and increasing scientific rigor in the form of pre-registration, blinding, and sham controls. Finally, we introduce the critical components of minimum device specification reporting for repeatability of studies of this type.

Transverse Fracture of the Mouse Femur with Stabilizing Pin

Fracture repair is an essential function of the skeleton that cannot be reliably modeled in vitro. A mouse injury model is an efficient approach to test whether a gene, gene product or drug influences bone repair because murine bones recapitulate the stages observed during human fracture healing. When a mouse or human breaks a bone, an inflammatory response is initiated, and the periosteum, a stem cell niche surrounding the bone itself, is activated and expands. Cells residing in the periosteum then differentiate to form a vascularized soft callus. The transition from the soft callus to a hard callus occurs as the recruited skeletal progenitor cells differentiate into mineralizing cells, and the bridging of the fractured ends results in the bone union. The mineralized callus then undergoes remodeling to restore the original shape and structure of the healed bone. Fracture healing has been studied in mice using various injury models. Still, the best way to recapitulate this entire biological process is to break through the cross-section of a long bone that encompasses both cortices. This protocol describes how a stabilized, transverse femur fracture can be safely performed to assess healing in adult mice. A surgical protocol including detailed harvesting and imaging techniques to characterize the different stages of fracture healing is also provided.

MAK122: A Novel Drug Utilizing Innovative Fracture Site Targeting Technology to Improve Bone Healing

Megakaryocytes play a pivotal role in the bone fracture healing process through enhancing osteoblast proliferation, osteoclastogenesis, and angiogenesis. Current fracture repair therapies require direct implantation during surgery (BMP-2, grafts etc.), which has limitations. In order to address this, a novel drug, compound MAK122, was created with targeting technology that directs its actions to the fracture site without needing to be implanted during surgery, limiting undesirable offsite effects, increasing the quantity of drug at the fracture site, and allowing for non-invasive treatment following assessment of the natural healing process. Therefore, this study examined the ability of MAK122 to stimulate megakaryocytes and subsequent bone healing. To accomplish this, male mice on a C57BL/6 background underwent a surgically induced femoral fracture. Following surgery, the mice were injected daily for the first 7 days with either saline (vehicle) or MAK122. Mice were then euthanized 2, 3 and 4 weeks post-surgery. Fracture healing was assessed by standard and novel methodologies. Biweekly X-rays were evaluated and bone union was scored showing that MAK122 accelerated bone healing compared to controls. Ex vivo µCT analysis demonstrated that MAK122 increased callus volume and the percentage of mineralized callus tissue compared to vehicle treatment. Biomechanical testing showed that MAK122 treatment resulted in stronger repairs as compared to vehicle treated controls with nearly a 2-fold increase in twist to failure and toughness parameters. Additionally, histological assessment demonstrated accelerated remodeling in MAK122 treated femurs compared to those treated with saline. Taken together, these pre-clinical data suggest that MAK122 is capable of promoting an environment in which megakaryocytes can favorably influence bone remodeling mechanisms, expediting fracture repair in murine models. Though further pharmacokinetic, pharmacodynamic, and toxicology studies are required, MAK122 displays potential to serve as a state-of-the-art therapy for improving fracture healing in humans.

A new approach for initial callus growth during fracture healing in long bones

The incidence of bone fracture has become a major clinical problem on a worldwide scale. In the past two decades there has been an increase in the use of computational tools to analyse the bone fracture problem. In several works, various study cases have been analysed to compare human and animal bone fracture healing. Unfortunately, there are not many publications about computational advances in this field and the existing approaches to the problem are usually similar.In this context, the objective of this work is the application of a diffusion problem in the model of the bone fragments resulting from fracture, working together with a mesh-growing algorithm that allows free growth of the callus depending on the established conditions, without a pre-meshed domain. The diffusion problem concerns the different biological magnitudes controlling the callus growth, among which Mesenchymal Stem Cells and chondrocytes concentrations were chosen, together with Tumour Necrosis Factor α and Bone Morphogenetic Protein as the factors influencing the velocity in the callus formation. A Finite Element approach was used to solve the corresponding diffusion problems, obtaining the concentration values along the entire domain and allowing detecting the zones in which biological magnitudes reach the necessary thresholds for callus growth. The callus growth is guided by a geometrical algorithm which performs an additional mesh generation process (self-added mesh) at each step of the iterative procedure until complete callus formation.The proposed approach was applied to different types of diaphyseal femoral fractures treated by means of intramedullary nailing. Axisymmetric models based on triangular quadratic elements were used, obtaining results in good agreement with clinical evidence of these kinds of fractures.The algorithm proposed has the advantage of a natural callus growth, without the existence of a previous mesh that may affect the conditions and direction of growth. The approach is intended for the initial phase of callus growth. Future work will address the implementation of the corresponding formulations for tissue transformation and bone remodelling in order to achieve complete fracture healing.

Effect of weekly teriparatide injections on osteoporotic fracture healing: protocol for a double-blind, randomised controlled trial

IntroductionBoth animal studies and clinical trials have shown that daily parathyroid hormone administration promotes bone fracture healing. We previously found that weekly injections of the recombinant human parathyroid hormone teriparatide...

Type 1 diabetic Akita mice have low bone mass and impaired fracture healing.

Type 1 diabetes (T1DM) impairs bone formation and fracture healing in humans. Akita mice carry a mutation in one allele of the insulin-2 (Ins2) gene, which leads to pancreatic beta cell dysfunction and hyperglycemia by 5-6weeks age. We hypothesized that T1DM in Akita mice is associated with decreased bone mass, weaker bones, and impaired fracture healing. Ins2±(Akita) and wildtype (WT) males were subjected to femur fracture at 18-weeks age and healing assessed 3-21days post-fracture. Non-fractured left femurs were assessed for morphology (microCT) and strength (bending or torsion) at 19-21weeks age. Fractured right femurs were assessed for callus mechanics (torsion), morphology and composition (microCT and histology) and gene expression (qPCR). Both Akita and WT mice gained weight from 3 to 18weeks age, but Akita mice weighed less starting at 5weeks (-5.2%, p<0.05). At 18-20weeks age Akita mice had reduced serum osteocalcin (-30%), cortical bone area (-16%), and thickness (-17%) compared to WT, as well as reduced cancellous BV/TV (-39%), trabecular thickness (-23%) and vBMD (-31%). Mechanical testing of non-fractured femurs showed decreased structural (stiffness, ultimate load) and material (ultimate stress) properties of Akita bones. At 14 and 21days post fracture Akita mice had a significantly smaller callus than WT mice (~30%), with less cartilage and bone area. Assessment of torsional strength showed a weaker callus in Akita mice with lower stiffness (-42%), maximum torque (-44%) and work to fracture (-44%). In summary, cortical and cancellous bone mass were reduced in Akita mice, with lower bone mechanical properties. Fracture healing in Akita mice was impaired by T1DM, with a smaller, weaker fracture callus due to decreased cartilage and bone formation. In conclusion, the Akita mouse mimics some of the skeletal features of T1DM in humans, including osteopenia and impaired fracture healing, and may be useful to test interventions.

Effect of Osteoporosis Medication on Fracture Healing: An Evidence Based Review.

A systematic search was conducted and relevant studies that evaluated the influence of osteoporosis medications (bisphosphonates [BPs], denosumab, selective estrogen receptor modulators [SERMs], recombinant human parathyroid hormone teriparatide [TPTD], and strontium ranelate [SrR]) on wrist, hip, and spine fracture healing, were selected. BPs administration did not influence fracture healing and clinical outcomes after distal radius fracture (DRF). Similar results were observed in hip fracture, but evidence is lacking for spine fracture. Denosumab did not delay the non-vertebral fractures healing in one well-designed study. No studies evaluated the effect of SERMs on fracture healing in humans. One study reported shorter fracture healing times in TPTD treated DRF patients, which was not clinically meaningful. In hip fracture, recent studies reported better pain and functional outcomes in TPTD treated patients. However, in spine fracture, recent studies found no significant differences in fracture stability between TPTD treated patients and controls. Evidence is lacking for SrR, but it did not influence wrist fracture healing in one study. In comparisons between TPTD and BPs, fracture healing and physical scores were not significantly different in hip fracture by 1 study. In spine fracture, controversy exists for the role of each medication to the fracture stability, but several studies reported that fracture site pain was better in TPTD treated patients than BPs treated patients. Considering no clinical data of negative fracture healing of the antiresorptive medication and the danger of subsequent fracture after initial osteoporotic fracture, there is no evidence to delay initiation of osteoporosis medications after fracture.

Computational modeling of human bone fracture healing affected by different conditions of initial healing stage

BackgroundBone healing process includes four phases: inflammatory response, soft callus formation, hard callus development, and remodeling. Mechanobiological models have been used to investigate the role of various mechanical and biological factors on bone healing. However, the effects of initial healing phase, which includes the inflammatory stage, the granulation tissue formation, and the initial callus formation during the first few days post-fracture, are generally neglected in such studies.MethodsIn this study, we developed a finite-element-based model to simulate different levels of diffusion coefficient for mesenchymal stem cell (MSC) migration, Young’s modulus of granulation tissue, callus thickness and interfragmentary gap size to understand the modulatory effects of these initial phase parameters on bone healing.ResultsThe results quantified how faster MSC migration, stiffer granulation tissue, thicker callus, and smaller interfragmentary gap enhanced healing to some extent. However, after a certain threshold, a state of saturation was reached for MSC migration rate, granulation tissue stiffness, and callus thickness. Therefore, a parametric study was performed to verify that the callus formed at the initial phase, in agreement with experimental observations, has an ideal range of geometry and material properties to have the most efficient healing time.ConclusionsFindings from this paper quantified the effects of the initial healing phase on healing outcome to better understand the biological and mechanobiological mechanisms and their utilization in the design and optimization of treatment strategies. It is also demonstrated through a simulation that for fractures, where bone segments are in close proximity, callus development is not required. This finding is consistent with the concepts of primary and secondary bone healing.

Effects of Intermittent Administration of Parathyroid Hormone and Parathyroid Hormone-Related Protein on Fracture Healing: A Narrative Review of Animal and Human Studies.

ABSTRACTIntermittent administration of parathyroid hormone (PTH) stimulates skeletal remodeling and is a potent anabolic agent in bone. PTH‐related protein (PTHrP) is anabolic acting on the same PTH1 receptor and is in therapeutic use for osteoporosis. The body of literature for PTH actions in fracture healing is emerging with promising yet not entirely consistent results. The objective of this review was to perform a literature analysis to extract up‐to‐date knowledge on the effects of intermittent PTH and PTHrP therapy in bone fracture healing. A literature search of the PubMed database was performed. Clinical case studies and articles related to “regeneration,” “implant,” and “distraction osteogenesis” were excluded. A narrative review was performed to deliberate the therapeutic potential of intermittent PTH administration on fracture healing. A smaller number of studies centered on the use of PTHrP or a PTHrP analog were also reviewed. Animal studies clearly show that intermittent PTH therapy promotes fracture healing and revealed the strong therapeutic potential of PTH. Human subject studies were fewer and not as consistent as the animal studies yet provide insight into the potential of intermittent PTH administration on fracture healing. Differences in outcomes for animal and human studies appear to be attributed partly to variable doses, fracture sites, age, remodeling patterns, and bone architectures, although other factors are involved. Future studies to examine the dose, timing, and duration of PTH administration will be necessary to further delineate the therapeutic potential of PTH for fracture healing in humans. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

The impact of nonosteogenic factors on the expression of osteoprotegerin and RANKL during human fracture healing.

ObjectivesThe osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) balance is of the utmost importance in fracture healing. The aim of this study was therefore to investigate the impact of nonosteogenic factors on OPG and RANKL levels.MethodsSerum obtained from 51 patients with long bone fractures was collected over 48 weeks. The OPG and serum sRANKL (soluble RANKL) concentrations were measured using enzyme-linked immunosorbent assay (ELISA). Smoking habit, diabetes, and alcohol consumption were recorded.ResultsAge and sex greatly influenced preoperative serum levels of OPG and sRANKL but differences were even more pronounced during fracture healing. Statistical significance was observed for overall serum levels of OPG (p = 0.001) and sRANKL (p < 0.001) in older men and women (age greater than 50 years). Interestingly, OPG levels increased over time in older women but decreased over time in older men.ConclusionThese data suggest that nonosteogenic factors, most significantly age and sex, have a major impact on sRANKL and OPG levels. Given the established association of OPG and sRANKL levels and nonunion, these findings seem to be of clinical relevance.Cite this article: J. Starlinger, G. Kaiser, A. Thomas, K. Sarahrudi. The impact of nonosteogenic factors on the expression of osteoprotegerin and RANKL during human fracture healing. Bone Joint Res 2019;8:349–356. DOI: 10.1302/2046-3758.87.BJR-2018-0116.R3.

Influence of Menopause on Inflammatory Cytokines during Murine and Human Bone Fracture Healing.

Postmenopausal females display a chronic inflammatory phenotype with higher levels of circulating pro-inflammatory cytokines. Furthermore, the inflammatory response to injury may be altered under estrogen-deficiency, because it was shown previously that estrogen-deficient mice displayed increased levels of the inflammatory cytokines Midkine (Mdk) and Interleukin-6 (IL-6) in the early fracture hematoma. Because a balanced immune response to fracture is required for successful bone regeneration, this might contribute to the delayed fracture healing frequently observed in osteoporotic, postmenopausal fracture patients. In this study, we aimed to investigate whether further cytokines in addition to Mdk and IL-6 might be affected by estrogen-deficiency after fracture in mice and whether these cytokines are also relevant during human fracture healing. Additionally, we aimed to investigate whether serum from male vs. female fracture patients affects osteogenic differentiation of human mesenchymal stem cells (MSCs). To address these questions, female mice were either sham-operated or ovariectomized (OVX) and subjected to standardized femur osteotomy. A broad panel of pro- and anti-inflammatory cytokines was determined systemically and locally in the fracture hematoma. In a translational approach, serum was collected from healthy controls and patients with an isolated fracture. Mdk and IL-6 serum levels were determined at day 0, day 14 and day 42 after fracture. Subgroup analysis was performed to investigate differences between male and female fracture patients after menopause. In an in vitro approach, human MSCs were cultured with the collected patient serum and osteogenic differentiation was assessed by qPCR and alkaline-phosphatase staining. Our results suggest an important role for the pro-inflammatory cytokines Mdk and IL-6 in the response to fracture in estrogen-deficient mice among all of the measured inflammatory mediators. Notably, both cytokines were also significantly increased in the serum of patients after fracture. However, only Mdk serum levels differed significantly between male and female fracture patients after menopause. MSCs cultivated with serum from female fracture patients displayed significantly reduced osteogenic differentiation, which was attenuated by Mdk-antibody treatment. In conclusion, our study demonstrated increased Mdk levels after fracture in OVX mice and female fracture patients after menopause. Because Mdk is a negative regulator of bone formation, this might contribute to impaired osteoporotic fracture healing.

Serum proteomic assessment of the progression of fracture 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 <0.05). Ontology assessment associated these proteins with osteoblasts, monocyte/macrophage lineages, mesenchymal stem cell lines, hepatic tissues, and lymphocytes. Temporal clustering of these data identified proteins associated with inflammation, cartilage formation and bone remodeling stages of healing. VEGF, Wnt, and TGF-βsignaling pathways were restricted to the period of cartilage formation. Comparison of the proteomic and transcriptomic profiles showed that 87.5% of proteins in serum had concordant expression to their mRNA expression in the callus, while 12.5% of the protein and mRNA expression patterns were discordant. The discordant proteins that were elevated in the serum but down regulated in callus mRNA expression were related to clotting functions, allograft rejection, and complement function. While proteins down regulated in the serum and elevated in callus mRNA were associated with osteoblast function, NF-ĸb, and activin signaling. These data show the serum proteome may be used to monitor the different biological stages of fracture healing and have translational potential in assessing human fracture healing. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1153-1163, 2018.

No effect of teriparatide on migration in total knee replacement

Background and purpose — Aseptic loosening is a main cause of late revision in total knee replacement (TKR). Teriparatide, a recombinant parathyroid hormone (PTH), stimulates osteoblasts and has been suggested to improve cancellous bone healing in humans. This might also be relevant for prosthesis fixation. We used radiostereometric analysis (RSA) to investigate whether teriparatide influences prosthesis fixation. Early migration as measured by RSA can predict future loosening.Patients and methods — In a randomized controlled trial with blind evaluation, 50 patients with osteoarthritis of the knee were allocated to a teriparatide treatment group (Forsteo, 20 μg daily for 2 months postoperatively) or to an untreated control group. RSA was performed postoperatively and at 6 months, 12 months, and 24 months. The primary effect variable was maximal total point motion (MTPM) from 12 to 24 months.Results — Median maximal total point motion from 12 to 24 months was similar in the 2 groups (teriparatide: 0.14 mm, 10% and 90% percentiles: 0.08 and 0.24; control: 0.13 mm, 10% and 90% percentiles: 0.09 and 0.21). [Authors: this is perhaps better than using "10th" and "90th", which looks ugly in print./language editor] The 95% confidence interval for the difference between group means was −0.03 to 0.04 mm, indicating that no difference occurred.Interpretation — We found no effect of teriparatide on migration in total knee replacement. Other trials using the same dosing have suggested a positive effect of teriparatide on human cancellous fracture healing. Thus, the lack of effect on migration may have been due to something other than the dose. In a similar study in this issue of Acta Orthopaedica, we found that migration could be reduced with denosumab (Ledin et al. 2017). The difference in response between the anabolic substance teriparatide and the antiresorptive denosumab suggests that resorption has a more important role during the postoperative course than any deficit in bone formation.