Predicting fracture healing with blood biomarkers: the potential to assess patient risk of fracture nonunion.

Fracture non-union is a significant orthopaedic problem affecting a substantial number of patients yearly. Treatment of nonunions is devastating to patients and costly to the healthcare system. Unfortunately, the diagnosis of non-union is typically made in a reactionary fashion by an orthopaedic surgeon based on clinical assessment and radiographic features several months into treatment. For this reason, investigators have been trying to develop prediction algorithms; however, these have relied on population-based approaches and lack the predictive capability necessary to make individual treatment decisions. There is also a growing body of literature focussed on identifying blood biomarkers that are associated with non-union. This review describes the research that has been done in this area. Further studies of patient-centered, precision medicine approaches will likely improve fracture non-union diagnostic/prognostic capabilities.

Bioinformatic analysis and experimental identification of blood biomarkers for chronic nonunion.

BACKGROUND: Incomplete fracture healing may lead to chronic nonunion; thus, determining fracture healing is the primary issue in the clinical treatment. However, there are no validated early diagnostic biomarkers for assessing chronic nonunion. In this study, bioinformatics analysis combined with an experimental verification strategy was used to identify blood biomarkers for chronic nonunion. METHODS: First, differentially expressed genes in chronic nonunion were identified by microarray data analysis. Second, Dipsaci Radix (DR), a traditional Chinese medicine for fracture treatment, was used to screen the drug target genes. Third, the drug-disease network was determined, and biomarker genes were obtained. Finally, the potential blood biomarkers were verified by ELISA and qPCR methods. RESULTS: Fifty-five patients with open long bone fractures (39 healed and 16 nonunion) were enrolled in this study, and urgent surgical debridement and the severity of soft tissue injury had a significant effect on the prognosis of fracture. After the systems pharmacology analysis, six genes, including QPCT, CA1, LDHB, MMP9, UGCG, and HCAR2, were chosen for experimental validation. We found that all six genes in peripheral blood mononuclear cells (PBMCs) and serum were differentially expressed after injury, and five genes (QPCT, CA1, MMP9, UGCG, and HCAR2) were significantly lower in nonunion patients. Further, CA1, MMP9, and QPCT were markedly increased after DR treatment. CONCLUSION: CA1, MMP9, and QPCT are biomarkers of nonunion patients and DR treatment targets. However, HCAR2 and UGCG are biomarkers of nonunion patients but not DR treatment targets. Therefore, our findings may provide valuable information for nonunion diagnosis and DR treatment. TRIAL REGISTRATION: ISRCTN, ISRCTN13271153. Registered 05 April 2020-Retrospectively registered.

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.

Defective Proliferation and Osteogenic Potential with Altered Immunoregulatory phenotype of Native Bone marrow-Multipotential Stromal Cells in Atrophic Fracture Non-Union.

Bone marrow-Multipotential stromal cells (BM-MSCs) are increasingly used to treat complicated fracture healing e.g., non-union. Though, the quality of these autologous cells is not well characterized. We aimed to evaluate bone healing-related capacities of non-union BM-MSCs. Iliac crest-BM was aspirated from long-bone fracture patients with normal healing (U) or non-united (NU). Uncultured (native) CD271highCD45low cells or passage-zero cultured BM-MSCs were analyzed for gene expression levels, and functional assays were conducted using culture-expanded BM-MSCs. Blood samples were analyzed for serum cytokine levels. Uncultured NU-CD271highCD45low cells significantly expressed fewer transcripts of growth factor receptors, EGFR, FGFR1, and FGRF2 than U cells. Significant fewer transcripts of alkaline phosphatase (ALPL), osteocalcin (BGLAP), osteonectin (SPARC) and osteopontin (SPP1) were detected in NU-CD271highCD45low cells. Additionally, immunoregulation-related markers were differentially expressed between NU- and U-CD271highCD45low cells. Interestingly, passage-zero NU BM-MSCs showed low expression of immunosuppressive mediators. However, culture-expanded NU and U BM-MSCs exhibited comparable proliferation, osteogenesis, and immunosuppression. Serum cytokine levels were found similar for NU and U groups. Collectively, native NU-BM-MSCs seemed to have low proliferative and osteogenic capacities; therefore, enhancing their quality should be considered for regenerative therapies. Further research on distorted immunoregulatory molecules expression in BM-MSCs could potentially benefit the prediction of complicated fracture healing.

Impaired bone healing following treatment of established nonunion correlates with serum cytokine expression.

Delayed union and nonunion are a significant concern in long bone fractures and spinal fusions. Treatment of nonunion often entails multiple revision surgeries that further increase the financial, physical, and emotional burden on patients. The optimal treatment strategy for nonunions remains unclear in many cases, and the risk of complications after revision procedures remains high. This is in part due to our limited understanding of the biological mechanisms that inhibit proper bone healing and lead to nonunion. And yet, few preclinical models directly investigate how healing is impacted after establishment of nonunion, with most instead primarily focusing on treatment immediately after a fresh bone injury. Here, we utilized a critical size femoral defect model in rats where treatment was delayed 8 weeks post-injury, at which time nonunion was established. In this study, acute and delayed treatments with bone morphogenetic protein-2 (BMP-2) were assessed. We found that delayed treatment resulted in decreased bone formation and reduced mechanical strength compared to acute treatment, even when BMP-2 dose was increased by 2.5 times the acute treatment dose. Interestingly, serum cytokine analysis at 12 weeks post-treatment revealed signs of chronic immune dysregulation after delayed treatment. In particular, non-responders (rats that did not exhibit defect bridging) demonstrated higher overall expression of inflammatory cytokines, including TNFα and IL-1ß, compared to responders. These findings suggest that re-establishing long-term immune homeostasis may be critical for successful bone healing, particularly after nonunion. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:299-307, 2019.

Chemokine analysis as a novel diagnostic modality in the early prediction of the outcome of non-union therapy: a matched pair analysis.

BACKGROUND: Despite the regenerative capability of skeletal tissue fracture, non-union is common. Treatment of non-unions remains challenging, and early determination of the outcome is impossible. Chemokines play an important role in promoting the formation of new bone and remodeling existing bone. Despite their importance regarding the regulation of bone biology, the potential of chemokines as biological markers reflecting osseous regeneration is unknown. The purpose of this study was to determine (1) if serum chemokine expression levels correlate with the outcome of non-union surgery and (2) if chemokine expression analysis can be used to identify patients at risk for treatment failure. METHODS: Non-union patients receiving surgical therapy in our institution between March 2012 and March 2014 were prospectively enrolled in a clinical observer study. Regular clinical and radiological follow-up was conducted for 12 months including collection of blood during the first 12 weeks. Based on the outcome, patients were declared as responders or non-responders to the therapy. To minimize biases, patients were matched (age, sex, body mass index (BMI)) and two groups of patients could be formed: responders (R, n = 10) and non-responders (NR, n = 10). Serum chemokine expression (CCL-2, CCL-3, CCL-4, CXCL-10, CCL-11, and interferon gamma (IFN-γ)) was analyzed using Luminex assays. Data was compared and correlated to the outcome. RESULTS: CCL-3 expression in NR was significantly higher during the course of the study compared to R (p = 0.002), and the expression pattern of CCL-4 correlated with CCL-3 in both groups (NR: p < 0.001 and r = 0.63). IFN-γ expression in NR was continuously higher than in R (p < 0.001), and utilization of CCL-3 and IFN-γ serum expression levels 2 weeks after the treatment resulted in a predictive model that had an AUC of 0.92 (CI 0.74-1.00). CONCLUSION: Serum chemokine expression analysis over time is a valid and promising diagnostic tool. The chemokine expression pattern correlates with the outcome of the Masquelet therapy of lower limb non-unions. Utilization of the serum analysis of CCL-3 and IFN-γ 2 weeks after the treatment resulted in an early predictive value regarding the differentiation between patients that are likely to heal and those that are prone to high risk of treatment failure.

Evaluation of matrix metalloproteases as early biomarkers for bone regeneration during the applied Masquelet therapy for non-unions.

INTRODUCTION: In the current study, we sought to determine if serum concentrations of MMPs correlate with bone regeneration occurring during the course of the Masquelet-therapy and to identify if MMPs may serve as early biomarkers reflecting successful bone regeneration and tissue remodeling. MATERIAL AND METHODS: This study was designed as a prospective clinical observer study. We compared serum samples over the time of treatment, as a matched-pair analysis, from 10 patients who were treated successfully with the Masquelet-therapy (Responder) with 10 patients who did not respond to the Masquelet-therapy (Non-Responder). The quantitative measurement was performed with Luminex Performance Human High Sensitivity Assays according to manufacturer's instructions. The lab technician performing the Luminex assays was blinded to both patient data and clinical outcome. RESULTS: Analysis of the expression pattern of MMP-2, -8 and -9 showed significant differences between groups. Two days after the first step of the Masquelet therapy Responder showed peak values of MMP-8 and MMP-9 that where significantly higher (p = 0.003 and p = 0.042, respectively) than in Non-Responder. In contrast serum levels of MMP-2 were lower after the first step of the Masquelet therapy in the Non-Responder group. The ratio of MMP-9 and MMP-2 was significantly higher in the Responder group two days after step I (p = 0.031) as well as 4 weeks after step II (p = 0.030). CONCLUSION: The findings of the current study emphasize the potential role of MMPs as biomarkers in bone remodeling. In particular, a distinct expression of MMP-2 correlates with successful bone regeneration, whereas initial overexpression of MMP-2 serum levels might identify patients that have a higher risk for a poor outcome of the Masquelet-therapy. Furthermore, we were able to introduce the serum analysis of the ratio of MMP-9 and MMP-2 as promising novel modality for early prediction of the outcome of the Masquelet therapy. Further analysis of this ratio over time subsequent to the second step might serve as an early indicator of a favorable response to the induced membrane technique.

An increase in myeloid cells after severe injury is associated with normal fracture healing: a retrospective study of 62 patients with a femoral fracture.

Background and purpose-Nonunion is common in femoral fractures. Previous studies suggested that the systemic immune response after trauma can interfere with fracture healing. Therefore, we investigated whether there is a relation between peripheral blood cell counts and healing of femur fractures. Patients and methods-62 multi-trauma patients with a femoral fracture presenting at the University Medical Centre Utrecht between 2007 and 2013 were retrospectively included. Peripheral blood cell counts from hematological analyzers were recorded from the 1st through the 14th day of the hospital stay. Generalized estimating equations were used to compare outcome groups. Results-12 of the 62 patients developed nonunion of the femoral fracture. The peripheral blood-count curves of total leukocytes, neutrophils, monocytes, lymphocytes, and platelets were all statistically significantly lower in patients with nonunion, coinciding with significantly higher CRP levels during the first 2 weeks after trauma in these patients. Interpretation-Patients who developed femoral nonunion after major trauma demonstrated lower numbers of myeloid cells in the peripheral blood than patients with normal fracture healing. This absent rise of myeloid cells seems to be related to a more severe post-traumatic immune response.

Evaluating the Use of Serum Inflammatory Markers for Preoperative Diagnosis of Infection in Patients with Nonunions.

PURPOSE: The aim of this study is to evaluate the effectiveness of laboratory serum tests in the diagnosis of infected nonunion. METHODS: Forty-two patients suspected of having infected nonunion were investigated in the study. The serum levels of white blood-cell count (WBC), C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and interleukin-6 (IL-6) were measured. A positive diagnosis of infection was made on the basis of the positive culture results. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of each test were calculated. RESULTS: The sensitivity and specificity of CRP both were higher than IL-6: 60.0% versus 57.1% and 85.7% versus 57.1%, respectively. With one, two, three, and four positive tests, the predicted probabilities of infection were 66.7%, 90.9%, 100%, and 100%, respectively, but the number of patients who had three or four positive tests was small. CONCLUSIONS: The diagnostic utility of IL-6 is inferior to CRP and the finding conflicts with previous conclusions drawn from periprosthetic infections. Laboratory analysis of serum inflammatory markers alone is not an effective screening tool for patients suspected of having an infected nonunion.

Evaluation of bone turnover markers and serum minerals variations for predicting fracture healing versus non-union processes in adult sheep as a model for orthopedic research.

Bone turnover markers (BTMs) have been considered as an auxiliary method of following the fracture healing process and for early prediction of impaired bone healing. A better understanding of the potential of BTMs in this application could allow for earlier interventions and improved patient care. The aim of this study with a large animal experimental model was to assess the variation of bone formation markers - namely the total alkaline phosphatase (ALP) and its bone-specific isoform (BALP), serum concentration of intact osteocalcin (OC), N-terminal propeptide type III procollagen (PIIINP) and of bone resorption markers - namely tartrate resistant acid phosphatase (TRAP) and deoxypyridinoline crosslink (DPD) during the first stages of a normal fracture healing process and of a segmental critical size defect (CSD), which progresses to a non-union process. Thirty healthy female sheep (Portuguese Churra-da-Terra-Quente breed), approximately 4-years-old, were enrolled in this study. Jugular venous blood samples were collected pre-operatively and at 1, 2, 3, 4, 6, 8, 10 and 12 post-operative weeks. The animals of the CSD group showed significant lower serum levels of BALP, OC and significant higher serum PIIINP levels at early stages of the fracture healing process, compared with animals that progressed in a normal fracture healing process. Serum BALP, OC and PIIINP levels could be useful as non-invasive auxiliary tools with other complementary methods for predicting the outcome of traumatic bone fractures.

Local transplantation of bone marrow concentrated granulocytes precursors can cure without antibiotics infected nonunion of polytraumatic patients in absence of bone defect.

PURPOSE: Infected, long bone non-unions present a significant clinical challenge. New and alternative therapies are needed to address this problem. The purposes of this study were to compare the number of circulating granulocyte-macrophage colony-forming units (CFU-GM) in the peripheral blood of polytraumatic patients with infected tibial non-unions and in the peripheral blood of control patients with the hypothesis that their number was decreased in polytraumatic patients; and to treat their infection without antibiotics and with local transplantation of bone marrow concentrated granulocytes precursors. METHODS: Thirty (18 atrophic and 12 hyperthrophic ) infected tibial non-unions (without bone defect) that occurred after open fractures in polytraumatic patients were treated without antibiotics and with percutaneous injection of autologous bone marrow concentrate (BMC) containing granulocytes precursors (CFU-GM). CFU-GM progenitors were assessed in the bone marrow aspirate, peripheral blood, and fracture site of these patients. The number of these progenitors was compared with the CFU-GM progenitors of control patient samples (healthy donors matched for age and gender). Outcome measures were: timing of union, callus formation (radiographs and CT scan), and recurrence of clinical infection. RESULTS: As compared to control patients, the number of CFU GM derived colonies was lower at peripheral blood in patients with infected nonunions. The bone marrow graft injected in nonunions contained after concentration 42 621 ± 20 350 CFU-GM-derived colonies/cc. Healing and cure of infection was observed at six months for 25 patients and at one year follow up for 30 patients. At the median ten year follow-up (range: 5 to 15), only one patient had clinical recurrent infection after healing (between 6 months and last follow-up). CONCLUSION: The peripheral blood of these polytraumatic patients with infected nonunions had a remarkable decrease in CFU-GM-derived colonies as compared with normal controls. Local transplantation of concentrated CFU-GM-derived colonies aspirated from bone marrow allowed cure of infection and healing without antibiotics.

Quantification of TGF-ß1, PDGF and IGF-1 cytokine expression after fracture treatment vs. non-union therapy via masquelet.

INTRODUCTION: Biochemical processes during bone regeneration can be analysed via quantification of peripheral serum cytokine levels. To date, serum levels of cytokines in patients treated with masquelet technique and patients with normal bone healing have not been compared. This comparison is supposed to deliver novel insights into the process of bone regeneration. Our aim was to validate this established method in the monitoring of bone regeneration after non-union treatment in masquelet technique. MATERIALS AND METHODS: Between 04/2008 and 01/2014 three groups were recruited: G1 (10 patients) with long bone non-unions, treated successfully with masquelet therapy, G2 (6 patients) with unsuccessful masquelet therapy and G3 (10 patients) with long bone fractures and normal bone healing. Peripheral blood samples were collected over a period of six months following a standardised time pattern in combination with clinical and radiologic follow up. TGF-ß1, PDGF-AB and IGF-1 were measured using commercially available immunoassays. RESULTS: TGF-ß1 levels in G1 and G2 demonstrated a parallel and lower overall concentration over time compared to G3. G3 showed a significant TGF-ß1 peak 2 weeks after surgery compared to G1 (p=0.0054). PDGF-AB concentrations were always lower in G2 than in G1 and G3. G3 peaked at week 2 with a significant higher value than in G2 (p=0.0177). IGF-1 showed lower overall serum concentrations in G2 than in G1 and G3. G1 had a peak level during the fourth week of follow-up. Compared to G2 this peak was significant (p=0.0015). CONCLUSIONS: This study shows that successful bone regeneration via masquelet technique only partially imitates cytokine expression of physiological bone healing. High expressions of IGF-1 correspond to a successful masquelet therapy while TGF-ß seems to play a minor role. These results assume that objective analysis of an effective non-union therapy with cytokine expression analysis is possible even with a small number of patients.

Deficiency of inducible and endothelial nitric oxide synthase results in diminished bone formation and delayed union and nonunion development.

BACKGROUND: Between 5% and 10% of all fractures fail to heal adequately resulting in nonunion of the fracture fragments. This can significantly decrease a patient's quality of life and create associated psychosocial and socio-economic problems. Nitric oxide (NO) and nitric oxide synthases (NOS) have been found to be involved in fracture healing, but until now it is not known if disturbances in these mechanisms play a role in nonunion and delayed union development. In this study, we explored the role of endothelial and inducible NOS deficiency in a delayed union model in mice. MATERIALS AND METHODS: A 0.45mm femur osteotomy with periosteal cauterization followed by plate-screw osteosynthesis was performed in the left leg of 20-24week old wild type, Nos2(-/-) and Nos3(-/-) mice. Contralateral unfractured legs were used as a control. Callus volume was measured using micro-computed tomography (µCT) after 28 and 42days of fracture healing. Immuno histochemical myeloperoxidase (MPO) staining was performed on paraffin embedded sections to assess neutrophil influx in callus tissue and surrounding proximal and distal marrow cavities of the femur. After 7 and 28days of fracture healing, femurs were collected for amino acid and RNA analysis to study arginine-NO metabolism. RESULTS: With µCT, delayed union was observed in wild type animals, whereas in both Nos2(-/-) and Nos3(-/-) mice nonunion development was evident. Both knock-out strains also showed a significantly increased influx of MPO when compared with wild type mice. Concentrations of amino acids and expression of enzymes related to the arginine-NO metabolism were aberrant in NOS deficient mice when compared to contralateral control femurs and wild type samples. DISCUSSION AND CONCLUSION: In the present study we show for the first time that the absence of nitric oxide synthases results in a disturbed arginine-NO metabolism and inadequate fracture healing with the transition of delayed union into a nonunion in mice after a femur osteotomy. Based on these data we suggest that the arginine-NO metabolism may play a role in the prevention of delayed unions and nonunions.

Biological perspectives of delayed fracture healing.

Fracture healing is a complex biological process that requires interaction among a series of different cell types. Maintaining the appropriate temporal progression and spatial pattern is essential to achieve robust healing. We can temporally assess the biological phases via gene expression, protein analysis, histologically, or non-invasively using biomarkers as well as imaging techniques. However, determining what leads to normal versus abnormal healing is more challenging. Since the ultimate outcome of fracture healing is to restore the original functions of bone, assessment of fracture healing should include not only monitoring the restoration of structure and mechanical function, but also an evaluation of the restoration of normal bone biology. Currently few non-invasive measures of biological factors of healing exist; however, recent studies that have correlated non-invasive measures with fracture healing outcome in humans have shown that serum TGFbeta1 levels appear to be an indicator of healing versus non-healing. In the future, developing additional measures to assess biological healing will improve the reliability and permit us to assess stages of fracture healing. Additionally, new functional imaging technologies could prove useful for better understanding both normal fracture healing and predicting dysfunctional healing in human patients.

Effect of the blood HbA1c level on surgical treatment outcomes of diabetics with ankle fractures.

OBJECTIVE: To investigate whether blood haemoglobin A 1c (HbA1c) levels was predicative of diabetic patients' responsiveness to surgical treatment of ankle fractures. METHODS: The relationship between blood HbA1c levels and surgical treatment outcomes of 21 diabetic patients undergoing open reduction internal fixation (ORIF) for ankle fractures was analyzed with Pearson correlation testing and t testing. All patients were treated with ORIF using standard surgical techniques. Treatment outcomes were defined using radiological outcome, the American Orthopaedic Foot and Ankle Score (AOFAS) ankle-hindfoot scale score, surgical revision rate, and complication rate. RESULTS: HbA1c levels were found to have a statistically significant correlation with poor radiological outcomes (r = 0.547) and AOFAS ankle-hindfoot scores (r = -0.592). Additionally, though rates of poor radiological outcome, revision, and complication were high in the diabetic population as a whole, these rates were considerably higher among individuals with elevated HbA1c (≥6.5%) and considerably lower among individuals with lower HbA1c (<6.5%) levels. CONCLUSION: Blood HbA1c levels appear to be predictive of risk and complication rates in the surgical treatment outcomes of diabetic patients with ankle fractures.

Decreased pool of mesenchymal stem cells is associated with altered chemokines serum levels in atrophic nonunion fractures.

Nonunion fractures can cause severe dysfunction and are often difficult to treat mainly due to a poor understanding of their physiopathology. Although many aspects of impaired fracture healing have been extensively studied, little is known about the cellular and molecular mechanisms leading to atrophic nonunion. Therefore, the aim of the present study was to assess the pools and biological functions of bone marrow-derived mesenchymal stem cells (hMSCs) and circulating endothelial progenitor cells (EPCs) in atrophic nonunion patients compared to healthy subjects, and the systemic levels of growth factors involved in the recruitment, proliferation and differentiation of these cells. In nonunions, the pool of hMSCs was decreased and their proliferation delayed. However, once committed, hMSCs from nonunions were able to proliferate, differentiate into osteoblastic cells and mineralize in vitro as efficiently as hMSCs from healthy subjects. In parallel, we found altered serum levels of chemokines and growth factors involved in the chemotaxis and proliferation of hMSCs such as leptin, interleukin-6 (IL-6) and its soluble receptor, platelet-derived growth factor-BB (PDGF-BB), stem cell factor (SCF) and insulin-like growth factor-1 (IGF-1). Moreover, we showed that the number of EPCs and their regulating growth factors were not affected in nonunion patients. If nonunion is generally attributed to a vascular defect, our results also support a role for a systemic mesenchymal and osteogenic cell pool defect that might be related to alterations in systemic levels of factors implicated in their chemotaxis and proliferation.

Is the expression of Transforming Growth Factor-Beta1 after fracture of long bones solely influenced by the healing process?

PURPOSE: Circulating TGF-ß1 levels were found to be a predictor of delayed bone healing and non-union. We therefore aimed to investigate some factors that can influence the expression of TGF-ß1. The correlation between the expression of TGF-ß1 and the different socio-demographic parameters was analysed. METHODS: Fifty-one patients with long bone fractures were included in the study and divided into different groups according to their age, gender, cigarette smoking status, diabetes mellitus and regular alcohol intake. TGF-ß1 levels were analysed in patient's serum and different groups were retrospectively compared. RESULTS: Significantly lower TFG-ß1 serum concentrations were observed in non-smokers compared to smokers at week 8 after surgery. Significantly higher concentrations were found in male patients compared to females at week 24. Younger patients had significantly higher concentrations at week 24 after surgery compared to older patients. Concentrations were significantly higher in patients without diabetes compared to those with diabetes at six weeks after surgery. Patients with chronic alcohol abuse had significantly higher concentrations compared to those patients without chronic alcohol abuse. CONCLUSION: TGF-ß1 serum concentrations vary depending upon smoking status, age, gender, diabetes mellitus and chronic alcohol abuse at different times and therefore do not seem to be a reliable predictive marker as a single-point-in-time measurement for fracture healing.

Assessment of compromised fracture healing.

No standard criteria exist for diagnosing fracture nonunion, and studies suggest that assessment of fracture healing varies among orthopaedic surgeons. This variability can be problematic in both clinical and orthopaedic trauma research settings. An understanding of risk factors for nonunion and of diagnostic tests used to assess fracture healing can facilitate a systematic approach to evaluation and management. Risk factors for nonunion include medical comorbidities, age, and the characteristics of the injury. The method of fracture management also influences healing. Comprehensive evaluation includes an assessment of the patient's symptoms, signs, and immune and endocrine status as well as the biologic capacity of the fracture, presence of infection, and quality of reduction and fixation. Diagnostic tests include plain radiography, CT, ultrasonography, fluoroscopy, bone scan, MRI, and several laboratory tests, including assays for bone turnover markers in the peripheral circulation. A systematic approach to evaluating fracture union can help surgeons determine the timing and nature of interventions.

Strongly enhanced levels of sclerostin during human fracture healing.

Sclerostin (SOST), an antagonist of Wnt signaling, is an important negative regulator of bone formation. However, no data on the role of SOST in the human fracture healing have been published so far. This study addressed this issue. Seventy-five patients with long bone fractures were included into the study and divided in two groups. The first group contained 69 patients with normal fracture healing. Six patients with impaired fracture healing formed the second group. Thirty-four volunteers donated blood samples as control. Serum samples were collected over a period of 1 year following a standardized time schedule. In addition, SOST levels were measured in fracture hematoma and serum of 16 patients with bone fractures. Fracture hematoma contained significantly higher SOST concentrations compared to patient's serum. SOST levels in fracture hematoma and in patient's serum were both significantly higher than in the serum of controls. Highly elevated SOST serum concentrations were found in patients with physiological fracture healing. SOST levels were decreased in patients with impaired fracture healing. However, this difference was not statistically significant. This is the first study to provide evidence of strongly enhanced SOST levels in patients with bone fracture. The results indicate local and systemic involvement of SOST in humans during fracture healing.

VEGF serum concentrations in patients with long bone fractures: a comparison between impaired and normal fracture healing.

Vascular endothelial growth factor (VEGF) plays an important role in the bone repair process as a potent mediator of angiogenesis and it influences directly osteoblast differentiation. Inhibiting VEGF suppresses angiogenesis and callus mineralization in animals. However, no data exist so far on systemic expression of VEGF with regard to delayed or failed fracture healing in humans. One hundred fourteen patients with long bone fractures were included in the study. Serum samples were collected over a period of 6 months following a standardized time schedule. VEGF serum concentrations were measured. Patients were assigned to one of two groups according to their course of fracture healing. The first group contained 103 patients with physiological fracture healing. Eleven patients with delayed or nonunions formed the second group of the study. In addition, 33 healthy volunteers served as controls. An increase of VEGF serum concentration within the first 2 weeks after fracture in both groups with a following decrease within 6 months after trauma was observed. Serum VEGF concentrations in patients with impaired fracture healing were higher compared to the patients with physiological healing during the entire observation period. However, statistically significant differences were not observed at any time point between both groups. VEGF concentrations in both groups were significantly higher than those in controls. The present results show significantly elevated serum concentrations of VEGF in patients after fracture of long bones especially at the initial healing phase, indicating the importance of VEGF in the process of fracture healing in humans.