Efficacy and safety of autologous or allogeneic mesenchymal stromal cells from adult adipose tissue expanded and combined with tricalcium phosphate biomaterial for the surgical treatment of atrophic nonunion of long bones: a phase II clinical trial.

BACKGROUND: Autologous bone grafting is the standard treatment for the surgical management of atrophic nonunion of long bones. Other solutions, such as bone marrow mesenchymal stem cells (BM-MSC) combined with phospho-calcium material, have also been used. Here we evaluate the safety and early efficacy of a novel procedure using autologous or allogenic adipose tissue mesenchymal stromal cells (AT-MSC) seeded in a patented tricalcium phosphate-based biomaterial for the treatment of bone regeneration in cases of atrophic nonunion. METHODS: This was a prospective, multicentric, open-label, phase 2 clinical trial of patients with atrophic nonunion of long bones. Biografts of autologous or allogenic AT-MSC combined with a phosphate substrate were manufactured prior to the surgical procedures. The primary efficacy was measured 6 months after surgery, but patients were followed for 12 months after surgery and a further year out of the scope of the study. All adverse events were recorded. This cohort was compared with a historical cohort of 14 cases treated by the same research team with autologous BM-MSC. RESULTS: A total of 12 patients with atrophic nonunion of long bones were included. The mean (SD) age was 41.2 (12.1) years and 66.7% were men. Bone healing was achieved in 10 of the 12 cases (83%) treated with the AT-MSC biografts, a percentage of healing similar (11 of the 14 cases, 79%) to that achieved in patients treated with autologous BM-MSC. Overall, two adverse events, in the same patient, were considered related to the procedure. CONCLUSIONS: The results of this study suggest that AT-MSC biografts are safe for the treatment of bone regeneration in cases of atrophic nonunion and reach high healing rates. TRIAL REGISTRATION: Study registered with EUDRA-CT (2013-000930-37) and ClinicalTrials.gov (NCT02483364).

Suppression of DNMT2/3 by proinflammatory cytokines inhibits CtBP1/2-dependent genes to promote the occurrence of atrophic nonunion.

Failure of bone healing after fracture often results in nonunion, but the underlying mechanism of nonunion pathogenesis is poorly understood. Herein, we provide evidence to clarify that the inflammatory microenvironment of atrophic nonunion (AN) mice suppresses the expression levels of DNA methyltransferases 2 (DNMT2) and 3A (DNMT3a), preventing the methylation of CpG islands on the promoters of C-terminal binding protein 1/2 (CtBP1/2) and resulting in their overexpression. Increased CtBP1/2 acts as transcriptional corepressors that, along with histone acetyltransferase p300 and Runt-related transcription factor 2 (Runx2), suppress the expression levels of six genes involved in bone healing: BGLAP (bone gamma-carboxyglutamate protein), ALPL (alkaline phosphatase), SPP1 (secreted phosphoprotein 1), COL1A1 (collagen 1a1), IBSP (integrin binding sialoprotein), and MMP13 (matrix metallopeptidase 13). We also observe a similar phenomenon in osteoblast cells treated with proinflammatory cytokines or treated with a DNMT inhibitor (5-azacytidine). Forced expression of DNMT2/3a or blockage of CtBP1/2 with their inhibitors can reverse the expression levels of BGLAP/ALPL/SPP1/COL1A1/IBSP/MMP13 in the presence of proinflammatory cytokines. Administration of CtBP1/2 inhibitors in fractured mice can prevent the incidence of AN. Thus, we demonstrate that the downregulation of bone healing genes dependent on proinflammatory cytokines/DNMT2/3a/CtBP1/2-p300-Runx2 axis signaling plays a critical role in the pathogenesis of AN. Disruption of this signaling may represent a new therapeutic strategy to prevent AN incidence after bone fracture.

Accumulation of advanced glycation end products promotes atrophic nonunion incidence in mice through a CtBP1/2-dependent mechanism.

Atrophic nonunion (AN) is a complex and poorly understood pathological condition resulting from impaired fracture healing. Advanced glycation end products (AGEs) have been implicated in the pathogenesis of several bone disorders, including osteoporosis and osteoarthritis. However, the role of AGEs in the development of AN remains unclear. This study found that mice fed a high-AGE diet had a higher incidence of atrophic nonunion (AN) compared to mice fed a normal diet following tibial fractures. AGEs induced two C-terminal binding proteins (CtBPs), CtBP1 and CtBP2, which were necessary for the development of AN in response to AGE accumulation. Feeding a high-AGE diet after fracture surgery in CtBP1/2-/- and RAGE-/- (receptor of AGE) mice did not result in a significant occurrence of AN. Molecular investigation revealed that CtBP1 and CtBP2 formed a heterodimer that was recruited by histone deacetylase 1 (HDAC1) and runt-related transcription factor 2 (Runx2) to assemble a complex. The CtBP1/2-HDAC1-Runx2 complex was responsible for the downregulation of two classes of bone development and differentiation genes, including bone morphogenic proteins (BMPs) and matrix metalloproteinases (MMPs). These findings demonstrate that AGE accumulation promotes the incidence of AN in a CtBP1/2-dependent manner, possibly by modulating genes related to bone development and fracture healing. These results provide new insights into the pathogenesis of AN and suggest new therapeutic targets for its prevention and treatment.

Comparing outcomes of plate augmentation, nail exchange, and nail exchange with plate augmentation in the treatment of atrophic femoral shaft nonunion after intramedullary nailing: a multicenter retrospective study.

INTRODUCTION: Intramedullary (IM) nailing is the treatment of choice for femoral shaft fractures, but nonunion rates have been reported to be as high as 12%. Surgical interventions for nonunion involve exchange nailing or plate augmentation. Recently, a combined treatment of exchange nailing and plate augmentation has demonstrated good results, but its comparative effectiveness remains unclear. This study aimed to compare the clinical and radiographic outcomes of three different surgical interventions for atrophic femoral shaft nonunion, and investigate the factors that affect bone healing after reoperation. MATERIALS AND METHODS: A retrospective study was conducted at five university hospitals involving 149 patients with aseptic atrophic nonunion after IM nailing. These patients underwent reoperation with plate augmentation, exchange nailing, or combined treatment. Clinical and radiographic outcomes were assessed and compared according to reoperation procedure. Logistic regression analysis was performed to identify factors affecting persistent nonunion after reoperation. RESULTS: Of the cohort, 57 patients underwent plate augmentation, 64 underwent exchange nailing, and 28 received combined treatment. There were no significant differences in patient demographics among the groups. Exchange nailing produced a significantly lower union rate than did the combined treatment (82.8% vs. 100%, p = 0.016), whereas no significant difference was observed in the union rate and time to the union between plate augmentation and the combined treatment. Combined treatment showed the longest operative time and the greatest transfusion requirements. The risk factors for persistent nonunion included age, absence of autogenous bone grafts, and use of an exchange nailing technique. CONCLUSIONS: Exchange nailing as a treatment for atrophic femoral shaft nonunion after IM nailing resulted in a lower union rate. The efficacy of the combined treatment requires further study, and persistent nonunion may be influenced by age, bone grafting, and surgical techniques. A comprehensive approach targeting both biological environment and mechanical stability is crucial in the treatment of atrophic femoral shaft nonunion.

Bridge plating with decortication, autologous bone graft, and tight closure: a "stepwise surgical diamond concept" for treatment of nonunion in a series of fifty five patients.

PURPOSE: This study was conducted to assess a stepwise surgical procedure applied to treat a continuous series of patients with aseptic atrophic nonunion of long bones. METHODS: A retrospective review was performed of the medical files of patients treated by the senior author between January 2014 and January 2021 for aseptic atrophic nonunion of long bones using a standard stepwise surgical procedure consisting of four successive surgical steps: bridge locked plating, aggressive osteoperiosteal decortication, copious autologous iliac bone grafting, and tight closure without drainage. Patients were clinically and radiographically evaluated until bone healing, then at final follow-up for the purpose of the study. The primary objective of the study was to assess completion of bone healing; secondary objectives were the time required reaching bone union, the occurrence of complications at the iliac bone graft donor site, and the achievement of bone consolidation after a second attempt of treatment when indicated following failure of the index procedure. RESULTS: There were a total of 55 patients. One patient died from myocardial infarction before reaching bone healing and another one lost from early follow-up. There were remaining 53 patients with 37 years of mean age. The affected bone was the clavicle in five patients, humerus in 14, ulna in four, radius in one, femur in 13, and tibia in 16. The mean follow-up period was 3.4 years. A total of 52 patients (98.1%) achieved bone healing at a mean of 14.8 weeks from the index procedure. The only patient who did not reach bone healing after the index procedure was successfully revised using decortication-bone graft and new fixation with intra-medullary femoral nailing. Four patients (7.5%) developed local complications at the site of iliac bone harvesting. CONCLUSION: Our stepwise surgical procedure was very effective treating aseptic atrophic nonunion of long bones. However, as this study is a retrospective review of a limited series of one surgeon's experience, prospective comparative studies with large number of patients are suitable to define the advantages and indications of the procedure herein described.

Clinical and radiographic outcomes of revision with autogenous "structured" bone grafting combined with superior plate for recalcitrant atrophic nonunion of clavicular midshaft: a retrospective study.

PURPOSE: To assess the efficacy of autogenous "structured" bone grafting (ASBG), it was combined with superior plate (SP) revision operations for recalcitrant clavicular midshaft aseptic nonunion (CMAN). METHODS: This retrospective study included 12 patients who suffered from failure of autologous cancellous bone grafting (ACBG) and SP fixation because of CMAN. Visual analogue scale (VAS) data for pain and disabilities of arm, shoulder, and hand (DASH) scores of patients who underwent these procedures from January 2019 to December 2020, obtained before surgery and at the final follow-up time, were analysed. RESULTS: The average time between primitive fracture and this operative treatment was 29 months (15-38 months). The average duration of surgery was 153 minutes (range, 115-230 min), and the average blood loss was 560 ml (range, 350-860 ml). Complications occurred in two cases (16.67%): one was persistent pain at the donor site, and the other was a calf muscle vein thrombosis. No tissue infection was observed during follow-up. The mean follow-up time was 18 months (range, 12-30 months). All fractures progressed to osseous healing at a mean time of 14 weeks (range, 12-16 weeks). The mean pain VAS score significantly improved, from 4.8 ± 1.7 pre-operatively to 1.9 ± 1.1 at the final follow-up (P = 0.01). The mean DASH score improved significantly from 30.1 ± 11.2 pre-operatively to 7.8 ± 4. 2 at the final follow-up (P < 0.01). CONCLUSIONS: ASBG combined with SP revision surgery achieved excellent clinical outcomes in patients with recalcitrant CMAN.

Inflammatory processes and elevated osteoclast activity chaperon atrophic non-union establishment in a murine model.

BACKGROUND: Delayed bone healing, especially in long bones poses one of the biggest problems in orthopeadic and reconstructive surgery and causes tremendous costs every year. There is a need for exploring the causes in order to find an adequate therapy. Earlier investigations of human scaphoid non-union revealed an elevated osteoclast activity, accompanied by upregulated levels of TGF-beta and RANKL. Interestingly, scaphoid non-union seemed to be well vascularized. METHODS: In the current study, we used a murine femur-defect model to study atrophic non unions over a time-course of 10 weeks. Different time points were chosen, to gather insights into the dynamic processes of non-union establishment. RESULTS: Histological analyses as well as western blots and qRT-PCR indicated enhanced osteoclast activity throughout the observation period, paralleled by elevated levels of TGF-beta, TNF-alpha, MMP9, MMP13 and RANKL, especially during the early phases of non-union establishment. Interestingly, elevated levels of these mediators decreased markedly over a period of 10 weeks, as inflammatory reaction during non-union establishment seemed to wear out. To our surprise, osteoblastogenesis seemed to be unaffected during early stages of non-union establishment. CONCLUSION: Taken together, we gained first insights into the establishment process of atrophic non unions, in which inflammatory processes accompanied by highly elevated osteoclast activity seem to play a leading role.

Masquelet Reconstruction for Posttraumatic Segmental Bone Defects in the Forearm.

PURPOSE: The Masquelet technique is a procedure increasingly utilized for addressing segmental bone defects. The technique involves staged procedures consisting of bone debridement and temporary spacer placement to induce membrane formation, followed by delayed bone grafting. This report summarizes our center's experience with the Masquelet technique to reconstruct bone loss exclusively in the forearm. METHODS: We reviewed all cases in which the Masquelet technique was used to reconstruct segmental bone defects in the forearm resulting from acute trauma or nonunion, with or without infection, between 2014 and 2017 at a level-1 trauma center. Injury mechanism, prior surgeries, extent of bone defect, and demographic data were collected. Union was assessed along with treatment-related complications or reoperations. RESULTS: We identified 9 patients with segmental bony defects in the forearm treated with the Masquelet technique. Among this cohort, 5 patients had bone defects associated with acute open fractures and 4 patients presented with nonunion (1 atrophic and 3 infected nonunions). The median bony defect was 4.7 cm (range, 1.7-5.4 cm) at the time of grafting. Second stage grafting was performed with Reamer Irrigator Aspirator autograft from the femur in 8 patients and iliac crest bone cancellous graft in 1 patient. Union was achieved in all 9 patients. Six patients achieved union by 3-month follow-up, 2 patients by 6 months, and 1 patient by 12 months. One patient required a reoperation for plate fracture prior to union treated with revision internal fixation and grafting. CONCLUSIONS: The Masquelet technique effectively reconstructed traumatic and posttraumatic segmental defects in the forearm with a low incidence of complication. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic V.

Plate augmentation and hybrid bone grafting are effective treatments for atrophic nonunion of the femur with the original intramedullary nail retained in situ.

The purpose of this study is to evaluate the efficacy of plate augmentation and hybrid bone grafting for treating atrophic nonunion of the femur with original intramedullary nail retained in situ.In this study, 36 patients with atrophic nonunion of the femur who underwent surgery using the technique of plate augmentation and a hybrid bone grafting while retaining the original intramedullary nail in situ in Xi'an Honghui Hospital from January 2019 to December 2021 were enrolled. 28 patients who met the inclusion and exclusion criteria were ultimately included in the study. These 28 patients, consisting of 20 males and 8 females with a mean age of 38 years, were evaluated based on factors such as operation time, intraoperative blood loss, the average hospitalization days. Additionally, the results and function of these patients were evaluated by union time, Wu's scores of limb function and incidence of serious complications.All 28 patients achieved bone union at the 12 month follow-up, with an average follow-up time of 14.6 ± 4.2 months.The average operation time was 68.3 ± 11.2 min, and the average intraoperative blood loss was 140 ± 22.6 ml. Patients were hospitalized for an average of 5.8 ± 1.1 days. Full clinical and radiological bone union was achieved on average at 5.1 ± 1.9 months. The mean value of Wu's scores at the 12 month follow-up was significantly higher than before the operation. Limb function was excellent in 27 patients and good in one patient at the 12 month follow-up. However, five patients experienced the lower limb vein thrombosis, including one deep vein thrombosis and four lower limb intermuscular vein thromboses. One patient had a superficial infections of the surgical incision site, while three patients reported pain and numbness where their iliac bone graft was extracted at the 12 month follow-up. The technique of plate augmentation and hybrid bone grafting, combined with retaining the original intramedullary nail in situ has been shown to be a safe, effective, simply and standardizable practice for treating atrophic femoral nonunion with an intact original IMN fixation.

Mesenchymal stem cells derived from the fibrotic tissue of atrophic nonunion or the bone marrow of iliac crest: A donor-matched comparison.

Purpose: Atrophic nonunion is one of the most difficult complications of fracture. The cellular factors that contribute to atrophic nonunion are poorly understood, and mesenchymal stem cells (MSCs) are recognized as the key contributor to bone formation. This study aimed to characterize the MSCs isolated from the fibrotic tissue of atrophic nonunion (AN-MSCs) from the perspective of proliferation, differentiation potential, senescence, and paracrine function. Methods: Human atrophic fibrotic tissue was obtained from four donors aged 29-37 for isolating AN-MSCs, and donor-matched bone marrow acquired from the iliac crest for isolating MSCs (IC-MSCs) as control. The AN-MSCs or IC-MSCs in passage 3 were applied for the following evaluations. The surface markers expressed on the two cells were evaluated using flow cytometry. The proliferation of the two cells for up to 11 days was comparatively investigated. After osteogenic, chondrogenic, or adipogenic induction, multi-lineage differentiation of AN-MSCs or IC-MSCs was comparatively evaluated using lineage-specific stains and lineage-specific gene expression. Enzyme-linked immunosorbent assay (ELISA) assessment was applied to evaluate the paracrine function of AN-MSCs or IC-MSCs. Cellular senescence of AN-MSCs or IC-MSCs was evaluated using senescence-associated ß-galactosidase (SA-ß-gal) staining. Results: AN-MSCs or IC-MSCs from the four donors showed morphologic and immunophenotypic characteristics of MSCs, with the expression of MSCs markers and negative expression of hematopoietic markers. In general, AN-MSCs showed similar proliferation and adipogenic capacity with IC-MSCs. In contrast, IC-MSCs showed significantly higher osteogenic and chondrogenic capacity compared to AN-MSCs. Moreover, the culture medium of IC-MSCs contains significantly higher levels of VEGF, TGF-ß1, PDGF-BB, and IGF-1 than the culture medium of AN-MSCs. Lastly, the AN-MSCs are more prone to cellular senescence than the IC-MSCs. Conclusions: In-vitro, AN-MSCs were similar to IC-MSCs in proliferation and adipogenic capacity, but inferior to IC-MSCs in osteogenic and chondrogenic capacity, paracrine function, and anti-senescence.

Universal Long Bone Nonunion Classification.

Aim and background: The management of bone union disorders is a complex problem in orthopaedics, requiring a reliable and comprehensive classification system for accurate diagnosis and treatment. Despite advances in understanding pathophysiology, diagnosis, and treatment in this area, there is no generally accepted classification system. The aim of our work was to create a comprehensive classification, which will systemize the vast majority of bone union disorders, underline their differences and form the basis for their treatment. Methods: The key criteria for nonunion evaluation and treatment were identified based on the conducted literature review: Time from the initial event (delayed union or nonunion), location, type of pathology (A, Hypertrophic; B, Normotrophic; C, Oligotrophic) and the presence of hardware. Based on these criteria the ULBNC has been developed. Atrophic nonunions were excluded from this classification as they are considered segmental bone defects with special classification. Results: The ULBNC is based on the same principles of coding as the "gold standard" AO/OTA Fractures Classification system with alpha-numeric coding "from simple to complex." The choice of treatment method depends on the type, group, and subgroup of the nonunion as described. Conclusion: Universal Long Bone Nonunion Classification (ULBNC) is an alphanumeric system that describes the localization, type of pathology and morphologic characteristics of a nonunion. The use of ULBNC in practice and research will optimize and standardize the treatment of various types of bone healing disorders and eventually improve clinical outcomes. How to cite this article: Solomin LN, Semenistyy AA, Komarov AV, et al. Universal Long Bone Nonunion Classification. Strategies Trauma Limb Reconstr 2023;18(3):169-173.

Secondary injury and pro-inflammatory macrophages increase osteophyte growth and fracture healing in canine atrophic nonunion.

OBJECTIVES: In this study, we used a canine high-energy fracture model to examine the relationship between the early inflammatory reaction in adjacent tissues and the ability for osteophyte growth, aiming to identify causes that lead to atrophic nonunion inflammatory disease and to provide new strategies for prevention and treatment. MATERIALS AND METHODS: Forty-eight models of canine femoral high energy fractures were prepared and randomly divided into groups A and B (n=24 in each group). Dogs in both groups underwent open reduction and 6-hole plate internal fixation. Group A models were re-opened, and muscle near the bone was scraped at 14 d after the operation. On days 3, 17, 28, and 42 after fracture, 6 experimental dogs were euthanized per group, and the fracture specimens were used to examine pathologic changes and the growth of callus in the fractured end and its adjacent tissues. RESULTS: At day 14, neutrophil infiltration, with no macrophage recruitment, no mesenchymal cell proliferation, and no fracture healing cascade were observed in the adjacent tissues of both groups. Immediately after the second injury was performed in group A, many macrophages were seen, and mesenchymal cells proliferated, which initiated vigorous osteophyte growth and led to osteophyte healing. Atrophic nonunion was observed in group B without secondary injury. CONCLUSION: Macrophage recruitment deficiency in adjacent soft tissue in early surgery for high-energy fractures may be an important cause of atrophic nonunion. Secondary injury inflammation can effectively recruit mononuclear macrophages, generate osteoclasts, re-initiate the growth of osteophytes, and promote fracture healing.

Percutaneous injection of platelet-rich plasma to treat atrophic nonunion after internal fixation of ulnar fracture: a case report.

Non-union is a serious postoperative complication of fracture. Early detection and intervention can avoid revision surgery. Platelet-rich plasma releases many active tissue factors and has potential to promote fracture healing. Percutaneous injection of platelet-rich plasma at the fracture site may avoid surgical treatment when non-union occurs. We present a case of atrophic non-union of an ulna fracture treated conservatively with percutaneous injection of platelet-rich plasma.

Osteoperiosteal Decortication and Autogenous Cancellous Bone Graft Combined with Bridge Plating for Non-hypertrophic Diaphyseal Nonunion.

BACKGROUD: The aim of this study was to evaluate results of osteoperiosteal decortication and autogenous cancellous bone graft combined with a bridge plating technique in atrophic and oligotrophic femoral and tibial diaphyseal nonunion. METHODS: We retrospectively reviewed 31 patients with atrophic or oligotrophic femoral and tibial diaphyseal nonunion treated with osteoperiosteal decortication and autogenous cancellous bone graft between January 2008 and December 2018. Patients with hypertrophic nonunion, infected nonunion, and nonunion treated with autogenous cancellous bone graft alone were excluded. The nonunion site was exposed by using the Judet technique of osteoperiosteal decortication. Nonunion with a lack of stability was stabilized with a new plate using a bridge plating technique or augmented by supplemental fixation with a plate. Nonunion with malalignment was stabilized with a new plate after deformity correction. Autogenous cancellous bone graft was harvested from the posterior iliac crest and placed within the area of decortication. A basic demographic survey was conducted, and the type of existing implants, mechanical stability of the implants, the type of implants used for stabilization, the operation time, the time to bone union, and postoperative complications were investigated. RESULTS: The average follow-up period was 33.3 months (range, 8-108 months). The operation time was 207 minutes (range, 100-351 minutes). All but 1 nonunion (96.7%) were healed at an average of 4.2 months (range, 3-8 months). In 1 patient, bone union failed due to implant loosening with absorbed bone graft, and solid union was achieved by an additional surgery for stable fixation with a new plate, osteoperiosteal decortication, and autogenous cancellous bone graft. There were no other major complications such as neurovascular injuries, infection, loss of fixation, and malunion. CONCLUSIONS: Osteoperiosteal decortication and autogenous cancellous bone graft combined with stable fixation by bridge plating showed reliable outcomes in atrophic and oligotrophic diaphyseal nonunion. This treatment modality can be effective for treating atrophic and oligotrophic diaphyseal nonunion because it is very helpful stimulating bone union.

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

BACKGROUND AND AIM: The 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 METHODS: The 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. RESULTS: The 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. CONCLUSION: The 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.

miR-1323 suppresses bone mesenchymal stromal cell osteogenesis and fracture healing via inhibiting BMP4/SMAD4 signaling.

BACKGROUND: Atrophic non-union fractures show no radiological evidence of callus formation within 3 months of fracture. microRNA dysregulation may underlie the dysfunctional osteogenesis in atrophic non-union fractures. Here, we aimed to analyze miR-1323 expression in human atrophic non-union fractures and examine miR-1323's underlying mechanism of action in human mesenchymal stromal cells. METHODS: Human atrophic non-union and standard healing fracture specimens were examined using H&E and Alcian Blue staining, immunohistochemistry, qRT-PCR, immunoblotting, and ALP activity assays. The effects of miR-1323 mimics or inhibition on BMP4, SMAD4, osteogenesis-related proteins, ALP activity, and bone mineralization were analyzed in human mesenchymal stromal cells. Luciferase reporter assays were utilized to assay miR-1323's binding to the 3'UTRs of BMP4 and SMAD4. The effects of miR-1323, BMP4, and SMAD4 were analyzed by siRNA and overexpression vectors. A rat femur fracture model was established to analyze the in vivo effects of antagomiR-1323 treatment. RESULTS: miR-1323 was upregulated in human atrophic non-union fractures. Atrophic non-union was associated with downregulation of BMP4 and SMAD4 as well as the osteogenic markers ALP, collagen I, and RUNX2. In vitro, miR-1323 suppressed BMP4 and SMAD4 expression by binding to the 3'UTRs of BMP4 and SMAD4. Moreover, miR-1323's inhibition of BMP4 and SMAD4 inhibited mesenchymal stromal cell osteogenic differentiation via modulating the nuclear translocation of the transcriptional co-activator TAZ. In vivo, antagomiR-1323 therapy facilitated the healing of fractures in a rat model of femoral fracture. CONCLUSIONS: This evidence supports the miR-1323/BMP4 and miR-1323/SMAD4 axes as novel therapeutic targets for atrophic non-union fractures.

NSM00158 Specifically Disrupts the CtBP2-p300 Interaction to Reverse CtBP2-Mediated Transrepression and Prevent the Occurrence of Nonunion.

Carboxyl-terminal binding proteins (CtBPs) are transcription regulators that control gene expression in multiple cellular processes. Our recent findings indicated that overexpression of CtBP2 caused the repression of multiple bone development and differentiation genes, resulting in atrophic nonunion. Therefore, disrupting the CtBP2-associated transcriptional complex with small molecules may be an effective strategy to prevent nonunion. In the present study, we developed an in vitro screening system in yeast cells to identify small molecules capable of disrupting the CtBP2-p300 interaction. Herein, we focus our studies on revealing the in vitro and in vivo effects of a small molecule NSM00158, which showed the strongest inhibition of the CtBP2-p300 interaction in vitro. Our results indicated that NSM00158 could specifically disrupt CtBP2 function and cause the disassociation of the CtBP2-p300-Runx2 complex. The impairment of this complex led to failed binding of Runx2 to its downstream targets, causing their upregulation. Using a mouse fracture model, we evaluated the in vivo effect of NSM00158 on preventing nonunion. Consistent with the in vitro results, the NSM00158 treatment resulted in the upregulation of Runx2 downstream targets. Importantly, we found that the administration of NSM00158 could prevent the occurrence of nonunion. Our results suggest that NSM00158 represents a new potential compound to prevent the occurrence of nonunion by disrupting CtBP2 function and impairing the assembly of the CtBP2-p300-Runx2 transcriptional complex.

TNF-α modulation via Etanercept restores bone regeneration of atrophic non-unions.

Treatment of atrophic non-unions, especially in long bones is a challenging problem in orthopedic surgery due to the high revision and failure rate after surgical intervention. Subsequently, there is a certain need for a supportive treatment option besides surgical treatment. In our previous study we gained first insights into the dynamic processes of atrophic non-union formation and observed a prolonged inflammatory reaction with upregulated TNF-α levels and bone resorption. In this study we aimed to improve bone regeneration of atrophic non-unions via TNF-α modulation in a previously established murine femoral segmental defect model. Animals that developed atrophic non-unions of the femur after 5 and 10 weeks were treated systemically for 10 and 5 weeks with Etanercept, a soluble TNF-α antibody. µCT scans and histology revealed bony bridging of the fracture gap in the treatment group, while bone formation in control animals without treatment was not evident. Moreover, osteoclasts were markedly decreased via modulation of the RANKL/OPG axis due to Etanercept treatment. Additionally, immunomodulatory effects via Etanercept could be observed as further inflammatory agents, such as TGF-ß, IL6, MMP9 and 13 were decreased in both treatment groups. This study is the first showing beneficial effects of Etanercept treatment on bone regeneration of atrophic non-union formation. Moreover, the results of this study provide a new and promising therapeutic option which might reduce the failure rate of revision surgeries of atrophic non-unions.

Treatment of atrophic nonunion via autogenous ilium grafting assisted by vertical fixation of double plates: A case series of patients.

OBJECTIVE: To investigate the efficacy of the treatment of atrophic nonunion using structural autogenous ilium bone grafting in combination with vertical fixation of double plates. METHODS: This retrospective study analysed the clinical data from consecutive patients with atrophic nonunion who underwent autogenous ilium grafting in combination with double-plate vertical fixation. The injury type and the bone affected by nonunion, the duration of nonunion and the outcomes following surgery were recorded for all patients. RESULTS: The study enrolled 43 patients with atrophic nonunion of the upper and lower limbs: 17 patients with tibial nonunion, 21 with femoral nonunion, four with humeral nonunion and one with radial shaft nonunion. The mean duration of postoperative follow-up was 14.5 months (range, 8-28 months). A total of 43 of 43 patients (100%) achieved a healed nonunion fracture without the occurrence of complications such as infection, fracture of internal fixation or pain in the harvesting site. Comprehensive postoperative assessments of bone healing and function were observed to be good and/or excellent in all 43 patients. CONCLUSION: Structural autogenous ilium grafting used in combination with double-plate vertical fixation can provide a stable structural environment for near optimal bone healing in patients with atrophic nonunion.

[Ilizarov external fixation without bone graft for atrophic femoral shaft nonunion].

OBJECTIVE: To explore the effectiveness of Ilizarov external fixation without bone graft in the treatment of atrophic femoral shaft nonunion. METHODS: The clinical data of 12 patients with atrophic femoral shaft nonunion admitted between October 2010 and January 2017 were retrospectively analyzed. There were 8 males and 4 females, aged from 24 to 61 years, with an average age of 41.7 years. The nonunion sites located in the middle and upper femur in 7 cases and in the distal femur or supracondylar in 5 cases. The disease duration ranged from 1 to 9 years, with an average of 3.7 years. Previous operations ranged from 1 to 9 times, with an average of 2.8 times. The original fixator was removed, the fracture end of nonunion was debrided, and Ilizarov external fixator was installed. In patients with the length of bone defect less than 4 cm, direct compression fixation was performed during operation; in patients with limb shortening more than 2.5 cm, proximal femoral osteotomy and bone lengthening components were required to prepare limb lengthening after operation; all patients did not receive bone graft. The wearing time of external fixator, clinical bone healing time of nonunion fracture end, and complications were recorded. The effectiveness was evaluated by Paley's nonunion evaluation criteria. RESULTS: All patients were followed up 24-50 months, with an average of 30 months. Bony union was achieved in all 12 cases with a healing time of 6.0-23.5 months (mean, 11.5 months). The wearing time of external fixator ranged from 7 to 25 months, with an average of 13.5 months. At last follow-up, according to Paley's nonunion evaluation criteria, the results were excellent in 6 cases, good in 4 cases, and fair in 2 cases, with an excellent and good rate of 83.3%. Sagittal angulation deformity of femur more than 7° occurred in 4 cases, with no significant effect on knee extension function, and no special treatment such as osteotomy was performed. Two patients had shorter limbs (>2.5 cm) after operation and were replaced by high shoes; 4 patients with trans-knee fixation lost knee joint mobility of 10-30° after operation; 10 cases of needle tract infection occurred, of which 4 cases with infection and loosening of fixed needle were replaced and re-fixed after needle extraction, the remaining 6 cases of infection without loosening of fixed needle were controlled by local dressing change, needle nursing, and oral cephalosporin anti-inflammatory drugs. No complications such as deep infection and vascular nerve injury occurred. CONCLUSION: Ilizarov external fixation has a high healing rate for atrophic femoral shaft nonunion, which is relatively minimally invasive and can avoid bone grafting. Its preliminary effectiveness is exact, and it is also effective for patients who have experienced multiple failed operations. It is necessary to pay attention to the nursing and rehabilitation training after external fixation.