Nonunion Model Research Articles

Development of a novel atrophic non-union model in rabbits: A preliminary study

Background and aimThe currently available atrophic non-union models rely on wide segmental excision of bone diaphysis to impede the process of healing but lack resemblance to the clinical scenario. The present study focused on developing an in vivo model of atrophic non-union fracture in rabbit radius that can replicate the clinical scenario. Materials and methodsThe atrophic non-union fracture model was developed by creating a 10 mm segmental bone defect in the radial diaphysis of five adult New Zealand White rabbits. The periosteum (2 mm) of the cut bone ends was cauterized using electrocautery to induce atrophy. Atrophic non-union was confirmed using radiographic and histologic evaluations on 30th postoperative day. ResultsThe radiographic signs of healing were completely absent in all the rabbits on 30th postoperative day, indicating inert bone ends. Histological findings further confirmed the presence of inert bone ends, indicating the development of atrophic non-union. ConclusionThe combination of the segmental bone defect, electrocautery induced thermal damage of bone end periosteum, and delayed treatment can induce the development of atrophic non-union fracture model in rabbits that can replicate the clinical scenario.

Targeting angiogenesis for fracture nonunion treatment in inflammatory disease

Atrophic fracture nonunion poses a significant clinical problem with limited therapeutic interventions. In this study, we developed a unique nonunion model with high clinical relevance using serum transfer-induced rheumatoid arthritis (RA). Arthritic mice displayed fracture nonunion with the absence of fracture callus, diminished angiogenesis and fibrotic scar tissue formation leading to the failure of biomechanical properties, representing the major manifestations of atrophic nonunion in the clinic. Mechanistically, we demonstrated that the angiogenesis defect observed in RA mice was due to the downregulation of SPP1 and CXCL12 in chondrocytes, as evidenced by the restoration of angiogenesis upon SPP1 and CXCL12 treatment in vitro. In this regard, we developed a biodegradable scaffold loaded with SPP1 and CXCL12, which displayed a beneficial effect on angiogenesis and fracture repair in mice despite the presence of inflammation. Hence, these findings strongly suggest that the sustained release of SPP1 and CXCL12 represents an effective therapeutic approach to treat impaired angiogenesis and fracture nonunion under inflammatory conditions.

Development of a tibial experimental non-union model in rats

BackgroundMany non-union animal models have been developed to explore the problems surrounding fracture healing. However, the existing models are not perfect and cannot satisfy all non-union studies. This study aimed to make a non-union model of the tibia in rats by cauterization of the posterior of 2 mm on both sides of the fracture end after open osteotomy of the tibia and fixing the fractured tibia with a Kirschner wire 0.8 mm in diameter.MethodsFor this study, 96 female adult Sprague-Dawley (SD) rats were used. The rats underwent surgery to produce a tibial open fracture and were fixed with a 0.8-mm diameter Kirschner wire. In 48 of the rats, the periosteum proximal and distal to the fracture end was cauterized.ResultsAt 2, 4, 6, and 8 weeks after surgery, radiological and histological analysis showed typical physiological healing in the control group, and the healing rate was 100% at 6 weeks. But the non-union group was characterized by resorption of the fracture ends with few callus formations and no bridging callus formation, and the healing rate was 0% at 8 weeks.ConclusionsThis method represents a reproducible model to create atrophic non-unions. This model provides a new option for studying the basic healing mechanisms and evaluating new therapies for bone regeneration and treatment of non-unions.

The risk assessment model of fracture nonunion after intramedullary nailing for subtrochanteric femur fracture.

To investigate the influencing factors of fracture nonunion after intramedullary nailing for subtrochanteric fractures and to construct a risk assessment model.Based on the multicenter retrospective analysis of 251 patients, all patients were divided into modeling group and verification group. In the modeling group, postoperative fracture nonunion rate, general data, fracture-related factors, surgical reduction-related factors, mechanical and biological factors were calculated, and the influencing factors of fracture nonunion were screened by univariate analysis. Logistic regression model was used for multifactor analysis to construct the risk assessment model. Based on the logistic regression model, the risk prediction model was constructed by drawing the Nomogram diagram. Through the verification group, the influencing factors were evaluated again, and the differentiation and calibration of the model were evaluated. The calibration degree was evaluated by Hosmer-Lemeshow test, goodness of fit test, and calibration curve. The discriminant degree was evaluated by the receiver operating characteristic curve.Fracture nonunion occurred in 34 of 149 patients in the modeling group. Among the 14 potential influencing factors, univariate analysis and logistic regression analysis showed that postoperative hip varus, intramedullary nail fixation failure, and reduction of fracture with large incision were the risk factors of fracture nonunion. The medial cortex fracture was seen reduced on X-Ray was a protective factor for fracture nonunion, and a regression equation was established. Based on the logistic regression model, the Nomogram diagram is drawn. Twenty-four cases of fracture nonunion occurred in the verification group. The area under the receiver operating characteristic curve was area under curve =0.883 > 0.7, indicating that there was a moderate differentiation to evaluate the occurrence of fracture nonunion after operation. The goodness of fit test: the Hosmers-Lemeshow test (X 2 = 2.921, P = .712 > .05) showed that the model had a good calibration.After intramedullary nailing of subtrochanteric fracture, hip varus, failure of intramedullary nail fixation and wide surgical dissection are the risk factors of fracture nonunion, and the postoperative reduction of medial cortex fracture is protective factor.National key research and development projects: 2016YFC0105806

Relational Exchange in Non-union Firms: A Configurational Framework for Workplace Dispute Resolution and Voice

For much of the 20th century, a sizeable proportion of the workforce in the United States had access to a combination of dispute resolution and voice options through the union grievance process. The vast majority of today’s workforce, however, no longer does. The focus of this article is the proliferation of alternative relational exchange models developed in non-union firms. The author develops a theoretical framework proposing variation in the overarching non-union models employed by firms as a function of distinct organizational features and strategies. These models are the product of distinct configurations of voice and dispute-resolution strategies. The author proposes five alternative non-union models, discusses the internal and external characteristics associated with them, and evaluates distinct employer and worker outcomes.

Prediction of Nonunion After Nonoperative Treatment of a Proximal Humeral Fracture.

The prevalence of nonunion after a proximal humeral fracture (PHF) and the risk factors for its occurrence are poorly defined. We aimed to estimate the rate of nonunion in nonoperatively treated patients and to produce a clinical model for its prediction. Two thousand two hundred and thirty adult patients (median age, 72 years [range, 18 to 103 years]; 75.5% were female) with a PHF underwent assessment of fracture union using standard clinical evaluation and conventional radiographs. We assessed the prevalence of nonunion and measured the effect of 19 parameters on healing. Best statistical practices were used to construct a multivariate logistic regression model. The PHF assessment of risk of nonunion model (PHARON) was externally validated in a subsequent prospectively collected population of 735 patients, treated by the same protocol in our institution. Overall, 231 (10.4%) of 2,230 patients developed nonunion. Only 3 (0.8%) of 395 patients with a head-shaft angle (HSA) of >140° developed nonunion; in this cohort, none of the measured candidate variables were independently predictive of nonunion on multivariate logistic regression analysis. In the larger cohort of 1,835 patients with an HSA of ≤140°, 228 (12.4%) developed nonunion. Decreasing HSA, increasing head-shaft translation (HST), and smoking were independently predictive of nonunion on multivariate analysis. The prevalence of nonunion was very low (1%) in the majority with both an HSA of >90° and HST of <50%, whereas the risk was much higher (83.7%) in the 8.3% with an HSA of ≤90° and HST of ≥50%. In both groups, the prevalence of nonunion was much higher in smokers. The prevalence of nonunion after PHF is higher than previously reported. Most patients have favorable risk-factor estimates and a very low risk of this complication, but a smaller subgroup is at much higher risk. The risk can be accurately estimated with PHARON, using standard clinical assessment tools. Prognostic Level III. See Instructions for Authors for a complete description of levels of evidence.

Single Versus Dual Headless Compression Screw Fixation of Scaphoid Nonunions: A Biomechanical Comparison.

Management of scaphoid nonunions with bone loss varies substantially. Commonly, internal fixation consists of a single headless compression screw. Recently, some authors have reported on the theoretical benefits of dual-screw fixation. We hypothesized that using 2 headless compression screws would impart improved stiffness over a single-screw construct. Using a cadaveric model, we compared biomechanical characteristics of a single tapered 3.5- to 3.6-mm headless compression screw with 2 tapered 2.5- to 2.8-mm headless compression screws in a scaphoid waist nonunion model. The primary outcome measurement was construct stiffness. Secondary outcome measurements included load at 1 and 2 mm of displacement, load to failure for each specimen, and qualitative assessment of mode of failure. Stiffness during load to failure was not significantly different between single- and double-screw configurations (P = .8). Load to failure demonstrated no statistically significant difference between single- and double-screw configurations. Using a qualitative assessment, the double-screw construct maintained rotational stability more than the single-screw construct (P = .029). Single- and double-screw fixation constructs in a cadaveric scaphoid nonunion model demonstrate similar construct stiffness, load to failure, and load to 1- and 2-mm displacement. Modes of failure may differ between constructs and represent an area for further study. The theoretical benefit of dual-screw fixation should be weighed against the morphologic limitations to placing 2 screws in a scaphoid nonunion.

How is Biodegradable Scaffold Effective in Gap Non-union? Insights from an Experiment.

To evaluate the role of composite (Chitosan/Chondroitin sulphate/gelatin/nano-bioglass) scaffold in the union of critical size bone defect created in the rabbit's ulna. The composite (Chitosan/Chondroitin sulphate/gelatin/nano-bioglass) scaffold was fabricated using the freeze-drying technique under standard laboratory conditions. The scaffold was cut into the appropriate size and transferred into the defect created (critical bone size defect 1cm) over the right ulna in the rabbit. The scaffold was not implanted on the left side thus the left side ulna served as control. Results were assessed on serial radiological examination. Rabbits were sacrificed at 20weeks for histopathological examination (Haematoxylin-Eosin staining and Mason's trichrome staining) and scanning electron microscope observation. Radiological scoring was done by Lane and Sandhu's scoring. Among 12 rabbits, 10 could complete the follow-up. Among those 10 rabbits, 8 among the test group showed good evidence of bone formation at the gap non-union scaffold implanted site. Histological evidence of new bone formation, collagen synthesis, scaffold resorption, minimal chondrogenesis was evident by 20weeks in the test group. Two rabbits had poor bone formation. The chitosan-chondroitin sulphate-gelatin-nano-bioglass composite scaffold is efficient in osteoconduction and osteoinduction in the gap non-union model as it is biocompatible, bioactive, and non-immunogenic as well.

Development and characterization of a predictive microCT-based non-union model in Fischer F344 rats.

Non-unions remain a clinical problem and are characterised by the failure to heal after a defined period of time. Current preclinical non-union models apply a wide variety of techniques to diminish intrinsic healing potential deviating from the clinical situation. The aim of this study was to develop and characterise a non-union model in rats using internal plate fixation without the need for additional healing insults, whereby bone healing can be longitudinally assessed using microCT. It was hypothesized that healing/non-unions can be accurately predicted at early time points by microCT. Female, skeletally mature Fischer F344 rats received a 2mm or 1mm femoral osteotomy, stabilized with either a 2mm thick plate or a 1.25mm thick plate. Healing was monitored by microCT over 14weeks and histological analysis at euthanasia. The mechanical environment was characterised using finite element (FE) modelling and biomechanical testing. The majority of animals receiving the 2mm thick plate displayed poor healing responses in both the 2mm and 1mm defect size groups. Bone and cartilage formation were markedly improved using the 1.25mm thick plate. MicroCT could accurately predict bone forming capacity at early time points (3-4weeks). The 2mm thick plating system confers poor healing responses in female Fischer F344 rats, comparable to atrophic non-unions. By reducing plate thickness to increase interfragmentary strain within the defect site healing is improved, leading to borderline healing situations or increased abundance of cartilage tissue present in the defect site with ultimate failure to bridge the defect (hypertrophic non-union). Furthermore, microCT can reliably identify delayed/non-healing animals within 4weeks, thereby allowing their selective targeting for the testing of novel, clinically relevant treatment strategies in different clinical situations aimed at restoring impaired bone healing.

The neurofibromatosis type I gene promotes autophagy via mTORC1 signalling pathway to enhance new bone formation after fracture.

Bone fracture is one of the most common injuries. Despite the high regenerative capacity of bones, failure of healing still occurs to near 10% of the patients. Herein, we aim to investigate the modulatory role of neurofibromatosis type I gene (NF1) to osteogenic differentiation of bone marrow–derived mesenchymal stem cells (BMSCs) and new bone formation after fracture in a rat model. We studied the NF1 gene expression in normal and non‐union bone fracture models. Then, we evaluated how NF1 overexpression modulated osteogenic differentiation of BMSCs, autophagy activity, mTORC1 signalling and osteoclastic bone resorption by qRT‐PCR, Western blot and immunostaining assays. Finally, we injected lentivirus‐NF1 (Lv‐NF1) to rat non‐union bone fracture model and analysed the bone formation process. The NF1 gene expression was significantly down‐regulated in non‐union bone fracture group, indicating NF1 is critical in bone healing process. In the NF1 overexpressing BMSCs, autophagy activity and osteogenic differentiation were significantly enhanced. Meanwhile, the NF1 overexpression inhibited mTORC1 signalling and osteoclastic bone resorption. In rat non‐union bone fracture model, the NF1 overexpression significantly promoted bone formation during fracture healing. In summary, we proved the NF1 gene is critical in non‐union bone healing, and NF1 overexpression promoted new bone formation after fracture by enhancing autophagy and inhibiting mTORC1 signalling. Our results may provide a novel therapeutic clue of promoting bone fracture healing.

Rotational Stability of Scaphoid Waist Nonunion Bone Graft and Fixation Techniques

Rotational instability of scaphoid fracture nonunions can lead to persistent nonunion. Wehypothesized that a hybrid Russe technique would provide improved rotational stability compared with an instrumented corticocancellous wedge graft in a cadaver model of scaphoid nonunion. A volar wedge osteotomy was created at the scaphoid waist in 16 scaphoids from matched-pair specimens. A wedge was inset at the osteotomy site or a 4× 16-mm strut was inserted in the scaphoid and a screw was placed along the central axis (model 1). The construct was cyclically loaded in torsion until failure. The screw was removed and the proximal and distal poles were debrided. A matching wedge and packed cancellous bone graft or an 8× 20-mm strut was shaped and fit inside the proximal and distal pole (model 2). A screw was placed and testing was repeated. In the first model, there was no significant difference in cycles to failure, target torque, or maximal torque between the strut graft and the wedge graft. Cycles to failure positively correlated with estimated bone density for the wedge graft, but not for the strut graft. In the second model, the strut graft had significantly higher cycles to failure, greater target torque, and higher maximal torque compared with the wedge graft. The number of cycles to failure was not correlated with estimated bone density for the wedge or the strut grafts. The hybrid Russe technique of inlay corticocancellous strut and screw fixation provides improved rotational stability compared with a wedge graft with screw fixation for a cadaver model of scaphoid waist nonunion with cystic change. The hybrid Russe technique may provide better rotational stability for scaphoid waist nonunions when the proximal or distal scaphoid pole is compromised, such as when there is extensive cystic change, when considerable debridement is necessary, or with revision nonunion surgery.

A new sequential animal model for infection-related non-unions with segmental bone defect

BackgroundThe treatment of fracture-related infections (FRI) is still a challenge for orthopedic surgeons. The prevalence of FRI is particularly high in open fractures with extensive soft-tissue damage. This study aimed to develop a new two-step animal model for non-unions with segmental bone defects, which could be used to evaluate new innovative bone substitutes to improve the therapeutic options in humans with FRI and bone defects.MethodsAfter randomization to infected or non-infected groups, 30 Sprague-Dawley rats underwent a transverse osteotomy of the mid-shaft femur with a 5 mm defect. Additionally, the periosteum at the fracture zone was cauterized at both sides. After intramedullary inoculation with 103 CFU Staphylococcus aureus (infected group) or PBS (non-infected group), a fracture stabilization was done by intramedullary K-wires. After 5 weeks, the bone healing process was evaluated, and revision surgery was performed in order to obtain increased bone healing. The initial K-wires were removed, and debridement of the osteotomy-gap was done followed by a more stable re-osteosynthesis with an angle-stable plate. After further 8 weeks all rats were euthanized and the bone consolidation was tested biomechanically and the callus formation quantitatively by micro-CT analysis.ResultsWe developed and presented a new two-stage non-union animal model through a targeted S. aureus infection. After 5 weeks, all animals showed a non-union irrespective of assignment to the infected and non-infected group. Lane and Sandhu score showed a higher callus formation in the infected group. In all infected animals, the inoculated S. aureus strain was detected in the revision surgery. The second surgery did not improve bone healing, as shown by the Lane Sandhu score and in the μ-CT analysis. Similarly, biomechanical testing showed in both groups a significantly lower maximum torque as compared to the contralateral side (p < 0.0001).ConclusionsWe were able to successfully develop a new two-stage non-union animal model, which reflects a genuine clinical situation of an infection-related non-union model with segmental bone defects. This model could be used to evaluate various therapeutic anti-infectious and osteoinductive strategies in FRIs.

Two new Yb(III)-based coordination polymers: structural characterization and treatment effect on femur fracture nonunion combined with PRP and n-HA/PA66

Two novel coordination polymers based on Yb(III) ion as the metal ions have been successfully prepared by using two C3-symmetrical ligands via a solvothermal synthesis approach, and their chemical formulae are [Yb1.5(TAPB)1.5(DMF)]·9DMF}n (1, TAPB = 4,4′,4″-(triazine-2,4,6-triyl-tris-(benzene-4,1-diyl)) tribenzoate) and [Yb(TCBPA)·DMF]n (2, H3TCBPA = tris(4′-carboxybiphenyl)amine). Furthermore, the two complexes were applicated for the fracture nonunion treatment combined with plate let rich plasma (PRP) and n-HA/Polyamide66 (n-HA/PA66). Firstly, the Rat Femur Fracture Nonunion Model was constructed, and compounds 1 or 2 was used for indicated treatment combined with PRP and n-HA/PA66, the enzyme linked immunosorbent assay detection was used for the osteocalcin concentration measurement. The reverse transcription-polymerase chain reaction was carried out to determine alp and Runx2 relative expression level.

Synthesis and Characterization of a Biocomposite Bone Bandage for Controlled Delivery of Bone-Active Drugs in Fracture Nonunions.

Fracture nonunions are common in orthopedics and their treatment often involves multiple surgical interventions. The aim of this study was to fabricate and characterize a gelatin-nano-hydroxyapatite membrane (GM)-based bone bandage for controlled delivery of bio-active molecules; recombinant human bone morphogenic protein-2 (rhBMP-2) and zoledronic acid (ZA) to promote osteoinduction and prevent callus resorption, respectively. In vitro cell-material interaction experiments using MC3T3 cells seeded on the GM indicated good biocompatibility. rhBMP-2-functionalized GM promoted osteogenic differentiation of MC3T3 cells and the rhBMP-2 bio-activity thus remained, as indicated by increased levels of alkaline phosphatase compared to only GM. The GM released a small amount (1.1%) of rhBMP-2 in vitro over a period of 5 weeks, demonstrating a strong interaction of rhBMP-2 with the GM. In the first animal study, the GM specimens loaded with rhBMP-2 or with the combination of rhBMP-2 + ZA were placed in the abdominal muscle pouch of rats. In the GM + rhBMP-2 + ZA group, significantly higher bone volume (21.5 ± 5.9 vs 2.7 ± 1.0 mm3) and area (3.3 ± 2.3 vs 1.0 ± 0.4 mm2) of bone were observed compared to GM + rhBMP-2 after 4 weeks, as indicated by micro-computed tomography and histomorphometry, respectively. Finally, a nonunion model in rats was used to evaluate the efficacy of the GM bandage and bio-active molecules in healing of fracture nonunions. The GM functionalized with rhBMP-2 + ZA led to higher bone formation around the fracture (63.9 ± 19.0 vs 31.8 ± 3.7 mm3) and stronger fracture callus (110.8 ± 46.8 vs 45.6 ± 17.8 N) compared to the empty controls. However, the overall union rate was only marginally improved. The GM alone or combined with ZA did not aid in bone healing in this model. Thus, this study shows that controlled delivery of rhBMP-2 + ZA via the developed GM is a promising approach that could aid in earlier full load bearing in patients with nonunion.

Robust method to create a standardized and reproducible atrophic non-union model in a rat femur

ObjectiveNo evidence exists about which biological approach is more reliable for creating non-union model. We investigated how to create a reproducible atrophic non-union model in a rat femur. MethodsWe compared three groups: simple osteotomy (group A), partial periosteum cauterization (group B), and extensive periosteum and bone marrow resection (group C). ResultsAll samples in group C demonstrated atrophic non-union in radiological, histological, and biomechanical analyses, however half of the samples in group B showed fracture healing at week 16. ConclusionExtensive resection of periosteum and bone marrow is important for a reproducible atrophic non-union model in a rat femur.

Exosomes from bone marrow mesenchymal stem cells enhance fracture healing through the promotion of osteogenesis and angiogenesis in a rat model of nonunion

BackgroundAs important players in cell-to-cell communication, exosomes (exo) are believed to play a similar role in promoting fracture healing. This study investigated whether exosomes derived from bone marrow mesenchymal stem cells (BMMSC-Exos) could improve fracture healing of nonunion.MethodsBMMSC-Exos were isolated and transplanted into the fracture site in a rat model of femoral nonunion (Exo group) every week. Moreover, equal volumes of phosphate-buffered saline (PBS) and exosome-depleted conditioned medium (CM-Exo) were injected into the femoral fracture sites of the rats in the control and CM-Exo groups. Bone healing processes were recorded and evaluated by radiographic methods on weeks 8, 14 and 20 after surgery. Osteogenesis and angiogenesis at the fracture sites were evaluated by radiographic and histological methods on postoperative week 20. The expression levels of osteogenesis- or angiogenesis-related genes were evaluated in vitro by western blotting and immunohistochemistry. The ability to internalize exosomes was assessed using the PKH26 assay. Altered proliferation and migration of human umbilical vein endothelial cells (HUVECs) and mouse embryo osteoblast precursor cells (MC3TE-E1s) treated with BMMSC-Exos were determined by utilizing EdU incorporation, immunofluorescence staining, and scratch wound assay. The angiogenesis ability of HUVECs was evaluated through tube formation assays. Finally, to explore the effect of exosomes in osteogenesis via the BMP-2/Smad1/RUNX2 signalling pathway, the BMP-2 inhibitors noggin and LDN193189 were utilized, and their subsequent effects were observed.ResultsBMMSC-Exos were observed to be spherical with a diameter of approximately 122 nm. CD9, CD63 and CD81 were expressed. Transplantation of BMMSC-Exos obviously enhanced osteogenesis, angiogenesis and bone healing processes in a rat model of femoral nonunion. BMMSC-Exos were taken up by HUVECs and MC3T3-E1 in vitro, and their proliferation and migration were also improved. Finally, experiments with BMP2 inhibitors confirmed that the BMP-2/Smad1/RUNX2 signalling pathway played an important role in the pro-osteogenesis induced by BMMSC-Exos and enhanced fracture healing of nonunion.ConclusionsOur findings suggest that transplantation of BMMSC-Exos exerts a critical effect on the treatment of nonunion by promoting osteogenesis and angiogenesis. This promoting effect might be ascribed to the activation of the BMP-2/Smad1/RUNX2 and the HIF-1α/VEGF signalling pathways.

Murine Aseptic Surgical Model of Femoral Atrophic Nonunion

Although bone repair is typically an efficient process, an inadequate healing response can occur, with approximately 5-20% of fractures developing nonunion. Even with improved healing strategies and external fixation devices, overall rate of nonunion has not been significantly reduced, particularly for atrophic nonunion. Atrophic nonunion is characterized by sparse or no callus formation and is difficult to treat clinically, resulting in long-term pain and functional limitation. Reliable preclinical models are needed to study the pathophysiology of atrophic nonunion to create better treatment options. The MouseNail kit (RISystem, Landquart, Switzerland) provides a highly standardized approach in which stabilized segmental bone defects are achieved through interlocked intramedullary nailing. However, reliably performing this surgery is technically challenging, particularly while maintaining strict asepsis. Skilled and aseptic surgical execution is important and necessary because it ensures optimal animal welfare and reproducibility. Therefore, the aim of this paper is to describe:•Novel modifications to the MouseNail kit that allow for: 1) a completely aseptic surgical environment, including description of a hanging limb orthopedic aseptic preparation and 2) a reduction in fracture gap size necessary for induction of atrophic nonunion.•Pre- to post-operative recommendations to facilitate successful performance of murine orthopedic survival surgery.

MiR-26a promotes fracture healing of nonunion rats possibly by targeting SOSTDC1 and further activating Wnt/β-catenin signaling pathway.

Nonunion is a serious complication after fracture due to its difficulty of self-healing. MicroRNA-26a (miR-26a) has been known to play a crucial role in bone metabolism. In this study, we established a rat nonunion model by removing periosteum, and found that miR-26a was significantly upregulated. Osteogenic differentiation of mesenchymal stem cells (MSCs) isolated from bone marrow transfected with miR-26a mimics was significantly enhanced, evidenced by increased calcium deposition and expression levels of alkaline phosphatase (ALP) and osteocalcin. Bioinformatics analysis suggested that sclerostin domain-containing 1 (SOSTDC1) may be a target of miR-26a, which was confirmed by dual-luciferase assay and western blot. Besides, miR-26a was used for nonunion rats. Delightfully, radiographs of nonunion rats with miR-26a mimics administration showed obvious new bone formation compared with nonhealing control. Hematoxylin-eosin and Masson staining assays revealed that osteogenesis capacity was greatly enhanced by miR-26a mimics' administration. In addition, miR-26a mimics could promote osteogenic differentiation in nonunion rats, evidenced by increased protein levels of ALP and osteocalcin, while SOSTDC1 was suppressed. The injection of miR-26a mimics also gave rise to phosphorylation of GSK3β and nuclear accumulation of β-catenin, which indicated the activation of canonical Wnt/β-catenin signaling. In conclusion, we demonstrated that miR-26a promoted fracture healing of rats with nonunion in vivo and osteogenic differentiation of MSCs in vitro, possibly by targeting SOSTDC1, and that Wnt/β-catenin signaling pathway was involved in this process.

VEGF-loaded mineral-coated microparticles improve bone repair and are associated with increased expression of epo and RUNX-2 in murine non-unions.

A poor vascular supply of the fracture gap is a key factor for the development of atrophic non-unions. Mineral-coated microparticles (MCM) represent a sophisticated carrier system for the delivery of vascular endothelial growth factor (VEGF). Hence, we investigated whether VEGF-loaded MCM improve bone repair in non-unions. For this purpose, we analyzed binding and release kinetics of MCM for VEGF in vitro. Moreover, we applied VEGF-loaded or -unloaded MCM in a murine non-union model in vivo and studied the process of bone healing by means of biomechanical, radiological, histomorphometric, and Western blot techniques. MCM-free non-unions served as controls. The binding efficiency of MCM for VEGF was 46 ± 3% and the release profile revealed an initial minor burst release followed by a sustained release over a 50-day study period, thus, mimicking the physiological expression profile of VEGF during bone healing. In vivo, bone defects treated with VEGF-loaded MCM exhibited a higher bending stiffness, a higher fraction of bone volume/tissue volume and a larger callus area on days 14 and 70 when compared to the other groups. Western blot analyses on day 14 revealed a higher expression of VEGF, erythropoietin (EPO), and runt-related transcription factor 2, but not of EPO-receptor in bone defects treated with VEGF-loaded MCM. These findings demonstrate that the use of MCM for VEGF delivery shows great potential due to the ability to maintain protein stability and functionality in vivo. Moreover, the application of VEGF-loaded MCM represent a promising strategy for the treatment of non-unions. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Periosteum-derived mesenchymal progenitor cells in engineered implants promote fracture healing in a critical-size defect rat model.

An attractive alternative to bone autografts is the use of autologous mesenchymal progenitor cells (MSCs) in combination with biomaterials. We compared the therapeutic potential of different sources of mesenchymal stem cells in combination with biomaterials in a bone nonunion model. A critical-size defect was created in Sprague-Dawley rats. Animals were divided into six groups, depending on the treatment to be applied: bone defect was left empty (CTL); treated with live bone allograft (LBA); hrBMP-2 in collagen scaffold (CSBMP2 ); acellular polycaprolactone scaffold (PCL group); PCL scaffold containing periosteum-derived MSCs (PCLPMSCs ) and PCL containing bone marrow-derived MSCs (PCLBMSCs ). To facilitate cell tracking, both MSCs and bone graft were isolated from green fluorescent protein (GFP)-transgenic rats. CTL group did not show any signs of healing during the radiological follow-up (n=6). In the LBA group, all the animals showed bone bridging (n=6) whereas in the CSBMP2 group, four out of six animals demonstrated healing. In PCL and PCLPMSCs groups, a reduced number of animals showed radiological healing, whereas no healing was detected in the PCLBMSCs group. Using microcomputed tomography, the bone volume filling the defect was quantified, showing significant new bone formation in the LBA, CSBMP2 , and PCLPMSCs groups when compared with the CTL group. At 10weeks, GFP positive cells were detected only in the LBA group and restricted to the outer cortical bone in close contact with the periosteum. Tracking of cellular implants demonstrated significant survival of the PMSCs when compared with BMSCs. In conclusion, PMSCs improve bone regeneration being suitable for mimetic autograft design.