Aim. Debridement, antibiotic, and implant retention (DAIR) is an accepted treatment of early and late acute Total Knee Arthroplasty (TKA) infections. DAIR failure may adversely affect the outcome of a subsequent two-stage exchange arthroplasty. Controversy exists on risk factors that can affect DAIR's results. The aim of the study is to review presurgical, intrasurgical and postsurgical variables that could affect DAIR's result. Method. A retrospective study of 27 DAIRs performed between 2015–2019 to treat late acute TKA infections was carried out. Patients were divided into two groups depending on DAIR's outcome [Healing (H) vs non-healing group (NH)] according on the Delphi-based multidisciplinary consensus criteria on success after treatment of periprosthetic joint infection. We reviewed presurgical variables, including epidemiological variables (Age, Sex, comorbidities, ASA, Charlson, BMI, alcohol dependency), prosthesis variables (prosthesis type, primary cause of operation, primary TKA surgery center), infection variables (concomitant infection, previous antibiotic treatment, c-reactive protein, synovial WBC count, synovial % PMN, pathogen), KLIC score and CRIME 80 score. Surgical variables such as surgery duration and type of surgery (elective vs urgent). Post-surgical variables like antibiotic treatment duration and destination at discharge. Normal distribution was assessed by Shapiro-Wilk test. Mann Whitney U test was used to compare the two independent sample variables. Chi-squared test was used for qualitative variables. P-value was established at 0.05 and statistical power at 80%. Results. Infection Healing was achieved in 63% of patients. In presurgical variables, alcohol dependency, hypertension, liver disease, previous surgery performed in another institution were more frequent in NH group (p< 0.05). KLIC score value equal or greater than 4 had a higher risk of surgical failure (p < 0.05). Regarding surgical variables, the healing group had more negative cultures than de non-healing one (p<0.05). Regarding post-surgical variables, long term antibiotic treatment (six months) achieved more healing after DAIR (p<0.05). Conclusions. Alcohol dependency, hypertension, liver disease and KLIC score values equal or greater than 4, may increases the risk of DAIR failure. Finally, we observed that the long-term antibiotic treatment (6 months) favors healing after DAIR

There is strong evidence to support the use of bisphosphonates in the prevention of osteoporotic fractures. There has, however, been growing concern that prolonged use of bisphosphonates can lead to the development of atypical femoral fractures and can protract healing time. We conducted a retrospective study looking at all femoral fractures between 2011–2013. Of 109 patients, 12 were diagnosed with atypical femoral fractures. The mean age of presentation was 69 (52–92). Five patients held no history of falls and presented with hip pain. The remaining seven sustained minor falls. Seven patients were on bisphosphonates on presentation. Bisphosphonates were discontinued in five cases and continued in two. Bisphosphonates commenced in one patient who subsequently developed second fracture. All fractures were managed with intramedullary nailing. Healing time was prolonged in all cases (mean healing time 7.3 months). Three patients needed further surgeries to achieve union. Overall, we observed that patients with prolonged bisphosphonate intake were more susceptible to atypical fractures with a delayed recovery time. Increasing awareness amongst medical professionals may aid timely diagnoses and subsequent referrals to orthopaedics. Recognition of these fractures may also permit early discontinuation of bisphosphonates, which may prevent future fractures and reduced healing times

An established rabbit model was used to preliminarily investigate the effect of acellular triphase, namely bone-cartilage-tendon, scaffold (ATS) sandwiched with autologous bone mesenchymal stem cells (BMSCs) sheets on tendon-bone interface healing. Bone, fibrocartilage and tendon tissue were harvested from the rabbits and sectioned into a book-type scaffold. The scaffolds were decellularized and their characterization was presented. BMSCs were isolated and co-cultured with the scaffolds to verify their cytocompatibility. BMSCs sheets were fabricated and inserted into the book page of the scaffold to construct an autologous BMSCs-sheets/book-type ATS complex. The complex was implated in the right knee of rabbits which operated standard partial patellectomy for TBI regeneration using Imaging, histological and biomechanical examinations. The bone, fibrocartilage and tendon tissue were sectioned into a book-type scaffold before decellularization. Then we decellularized the above tissue and mostly preserved their microstructure and composition of the natural extracellular matrix, including collagen and proteoglycan. After the physicochemical and biological properties of the book-type ATS were evaluated, autologous BMSCs sheets were inserted into the book page of the scaffold to construct an autologous BMSCs-sheets/book-type ATS implants for TBI regeneration. In addition, the ATS has the advantages of non-toxicity, suitable for cell adhesion and growth as well as low immunogenicity while co-cultured with the BMSCs. At the same time, different scaffolds has the ability to induce the osteogenic, chondrogenic and tenogenic differentiation of BMSCs by immunofluorescence, reverse transcription-polymerase chain reaction and western blot analysis. To determine the efficacy of the tissue-engineered implants for TBI regeneration, we transplanted it into a rabbit patella-patellar tendon (PPT) injury model, and the rabbits were sacrificed at postoperative week 8 or 16 for the radiological, histological, and mechanical evaluation. Radiologically, Synchrotron radiation micro-computed tomography (SR-μCT) showed that BMSCs/ATS group significantly increased bone area, BV/TV, trabecular thickness and trabecular number at the healing interface as compared with other groups at postoperative week 8 or 16. Histologically, the BMSCs/ATS group showed more woven bone, and a more robust fibrocartilaginous junction with a characteristic matrix rich in proteoglycans was seen at the PPT healing interface in comparison with other groups after 8 weeks. At week 16, the healing interface in 3 groups displayed better remodeling with respect to postoperative week 8. Healing and remodeling at the PPT junction were almost complete, with a resemblance to a healthy BTI consisting of the characteristic 4 zones in all groups. At last, we used biomechanical test as functional parameters to evaluate the quality of tendon-bone healing. Biomechanical testing indicated that BMSCs/ATS group showed significantly higher failure load and stiffness than other groups at postoperative week 8 and 16. The complex composed of acellular triphase, namely bone-cartilage-tendon, scaffold (ATS) sandwiched with autologous bone mesenchymal stem cells (BMSCs) sheets can simulate the gradient structure of tendon-bone interface, inducing stem cell directional differentiation, so as to promote patella-patellar tendon interface healing effectively after injury

INTRODUCTION. Appropriate, well characterized animal models remain essential for preclinical research. This study investigated a relevant animal model for cancellous bone defect healing. Three different defect diameters of fixed depth were compared in both skeletally immature and mature sheep. This ovine model allows for the placement of four confined cancellous defects per animal. METHODS. Defects were surgically created and placed in the cancellous bone of the medial distal femoral and proximal tibial epiphyses (See Figure 1). All defects were 25 mm deep, with defect diameters of 8, 11, and 14 mm selected for comparison. Defects sites were flushed with saline to remove any residual bone particulate. The skeletally immature and mature animals corresponded to 18 month old and 5 year old sheep respectively. Animals were euthanized at 4 weeks post-operatively to assess early healing. Harvested sites were graded radiographically. The percentage of new bone volume within the total defect volume (BV/TV) was quantified through histomorphometry and μ-CT bone morphometry. Separate regions of interest were constructed within the defect to assess differences in BV/TV between periosteal and deep bone healing. Defect sites were PMMA embedded, sectioned, and stained with basic fuschin and methylene blue for histological evaluation. RESULTS. The animals tolerated the surgery well, with no incidence of fractures within the four weeks. Healing of the defects progressed via endochondral ossification, with none of the defects being completely healed within the 4 week time point. Bone volume fraction (BV/TV) significantly decreased with an increasing defect diameter. Actual bone volume (BV), however, increased with defect diameter, suggesting a correlation between biological response and severity of injury. Three distinct healing regions were found to exist within the defect and along the axis of the defect, with significant differences detected in the BV/TV between adjacent regions. Histologically, the 5 year old animals appeared to have decreased osteoblast activity, and lower osteocyte density within the newly formed woven bone. On occasion, the defects were found to intersect the tibial growth plate in the 18 month old animals, with bone replacing the proliferating chondrocyte zone (See Figure 2). Additionally, the 14 mm defect was not able to be placed in the tibia of sheep due to the possibility of the defect entering the tibial intramedullary (IM) canal, and the lack of cancellous bone between the tibial plateau and IM canal. Both these issues considerably affect this model and should be avoided. CONCLUSION. The surgical placement of 11 mm diameter defects in the proximal tibial and distal femoral epiphyses of skeletally mature sheep presents a suitable large animal model to study early healing of cancellous bone defects. This refined model allows for the placement of four separate non-healing defects within a single sheep, and allows for the possibility to reduce animal numbers required to obtain information

Introduction

Non-union is an unfortunate outcome of the fracture healing process for some patients; with an estimated annual incidence of 17.4- 18.9 per 100,00. The management of these patients depicts a significant clinical challenge for surgeons and financial burden to health services. External ultrasound stimulation devices (Exogen^TM) have been highlighted as a novel non invasive therapy to achieve union in cases of delayed and non-union. The aim of the current study was to assess the rate of union in patients using Exogen^TM therapy for delayed fracture union in a district general hospital.

We hypothesised that diet-induced obesity (DIO) would result in inferior enthesis healing in a rat model of rotator cuff (RC) repair and that dietary intervention in the peri-operative period would improve enthesis healing.

A total of 78 male Sprague-Dawley rats were divided into three weight-matched groups from weaning and fed either: control diet (CD), high-fat diet (HFD), or HFD until surgery, then CD thereafter (HF-CD). After 12 weeks, the left supraspinatus tendon was detached, followed by immediate surgical repair. At 2 and 12 weeks post-surgery, animals were culled, and RCs harvested for biomechanical and histological evaluation. Body composition and metabolic markers were assessed via DEXA and plasma analyses, respectively.

DIO was established in the HFD and HF-CD groups before surgery and subsequently reversed in the HF-CD group after surgery. Histologically, the appearance of the repaired entheses was poorer in both the HFD and HF-CD groups compared with the CD group at 12 weeks after surgery, with semiquantitative scores of 6.2 (P<0.01), 4.98 (P<0.01), and 8.7 of 15, respectively. The repaired entheses in the HF-CD group had a significantly lower load to failure (P=0.03) at 12 weeks after surgery compared with the CD group, while the load to failure in the HFD group was low but not significantly different (P=0.10). Plasma leptin were negatively correlated with histology scores and load to failure at 12 weeks after surgery.

DIO impaired enthesis healing in this rat RC repair model, with inferior biomechanical and histological outcomes. Restoring normal weight with dietary change after surgery did not improve healing outcomes. Circulating levels of leptin significantly correlated with poor healing outcomes. This pre-clinical rodent model demonstrates that obesity is a potentially modifiable factor that impairs RC healing and increases the risk of failure after RC surgery.

Anterior cruciate ligament (ACL) injuries have been increasing, especially amongst adolescents. These injuries can increase the risk for early-onset knee osteoarthritis (OA). The consequences of late-stage knee OA include structural joint change, functional limitations and persistent pain. Interleukin-6 (IL-6) is a pro-inflammatory biomarker reflecting knee joint healing, and increasing evidence suggests that IL-6 may play a critical role in the development of pathological pain. The purpose of this study was to determine the relationship between subjective knee joint pain and function, and synovial fluid concentrations of the pro-inflammatory cytokine IL-6, in adolescents undergoing anterior cruciate ligament reconstruction surgery.

Seven youth (12-17 yrs.) undergoing anterior cruciate ligament (ACL) reconstruction surgery participated in this study. They completed the Pedi International Knee Documentation Committee (Pedi-IKDC) questionnaire on knee joint pain and function. At the time of their ACL reconstruction surgery, synovial fluid samples were collected through aspiration to dryness with a syringe without saline flushing. IL-6 levels in synovial fluid (sf) were measured using enzyme linked immunosorbent assay. Spearman's rho correlation coefficient was used to determine the correlation between IL-6 levels and scores from the Pedi-IKDC questionnaire.

There was a statistically significant correlation between sfIL-6 levels and the Pedi-IKDC Symptoms score (-.929, p=0.003). The correlations between sfIL-6 and Pedi-IKDC activity score (.546, p = .234) and between sfIL-6 and total Pedi-IKDC score (-.536, p = .215) were not statistically significant.

This is the first study to evaluate IL-6 as a biomarker of knee joint healing in an adolescent population, reported a very strong correlation (-.929, p=0.003) between IL-6 in knee joint synovial fluid and a subjective questionnaire on knee joint pain. These findings provide preliminary scientific evidence regarding the relationship between knee joint pain, as determined by a validated questionnaire and the inflammatory and healing status of the patient's knee. This study provides a basis and justification for future longitudinal research on biomarkers of knee joint healing in patients throughout their recovery and rehabilitation process. Incorporating physiological and psychosocial variables to current return-to-activity (RTA) criteria has the potential to improve decision making for adolescents following ACL reconstruction to reduce premature RTA thereby reducing the risk of re-injury and risk of early-onset knee OA in adolescents.

There is conjecture on the optimal timing to administer bisphosphonate therapy following operative fixation of low- trauma hip fractures. Factors include recommendations for early opportunistic commencement of osteoporosis treatment, and clinician concern regarding the effect of bisphosphonates on fracture healing. We performed a systematic review and meta-analysis to determine if early administration of bisphosphonate therapy within the first month post-operatively following proximal femur fracture fixation is associated with delay in fracture healing or rates of delayed or non-union.

We included randomised controlled trials examining fracture healing and union rates in adults with proximal femoral fractures undergoing osteosynthesis fixation methods and administered bisphosphonates within one month of operation with a control group. Data was pooled in meta-analyses where possible. The Cochrane Risk of Bias Tool and the GRADE approach were used to assess validity.

For the outcome of time to fracture union, meta-analysis of three studies (n= 233) found evidence for earlier average time to union for patients receiving early bisphosphonate intervention (MD = −1.06 weeks, 95% CI −2.01 – −0.12, I²= 8%). There was no evidence from two included studies comprising 718 patients of any difference in rates of delayed union (RR 0.61, 95% CI 0.25–1.46). Meta-analyses did not demonstrate a difference in outcomes of mortality, function, or pain.

We provide low-level evidence that there is no reduction in time to healing or delay in bony union for patients receiving bisphosphonates within one month of proximal femur fixation.

Introduction

Frame HI is the #Days for device removal/cm. IM Nail HI is less relevant (31–45 D/cm).

Albizzia HI was 33 D/cm (1991–2003). Patients felt fine approximately 1M after end of lengthening (EoL), resuming normal life and sports. This sometimes resulted in implants fractures (e.g. skying before bone fusion). Ideally, the full fusion should occur at the EoL. We decided to shorten the HI to reach this target, optimising all parameters.

Bone marrow concentrates are being used to augment soft tissue healing. However, only 0.01% of these cells meet the criteria of a mesenchymal stem cell (MSC), which likely accounts for the variability in reported results. Previous studies using an established rat rotator cuff repair model have demonstrated that bone marrow-derived MSCs had no effect on healing. In this study we evaluated the effect of purified human MSCs on rotator cuff healing in an athymic rat model. Hypothesis: Purified human MSCs added to the repair site will improve biomechanical strength and fibrocartilage formation of the healing tendon.

Fifty-two athymic rats underwent unilateral detachment and repair of the supraspinatus tendon with either fibrin glue (control) or fibrin glue with 106 hMSCs (experimental) applied at the repair site. Flow cytometry verified the stem cell phenotype of the cells as CD73+, CD90+, CD105+, CD14-, CD34- and CD45-. Rats were sacrificed at 2 and 4 weeks, with 10 used for biomechanical testing and 3 for histologic analysis from each group.

Biomechanical testing revealed a significant increase in failure load (11.5±2.4N vs. 8.5±2.4N, p=0.002) and stiffness (7.1±1.2 N/mm vs. 5.7±2.1 N/mm, p0.17).

These data demonstrate the potential for stem cells to augment tendon healing. This is the first study to use purified stem cells, rather than simple bone marrow concentrate. In the future, cell sorting techniques and culture expansion could be used to select and expand the small population of true stem cells in bone marrow. Furthermore, healing could potentially be improved with repeat cell injection at an additional post-operative time point.

Aim

The time to onset of symptoms after fracture fixation is still commonly used to classify fracture-related infections (FRI). Early infections (<2 weeks) can often be treated with debridement, systemic antibiotics, irrigation, and implant preservation (DAIR). Late infections (>10 weeks) typically require implant removal as mature, antibiotic-tolerant biofilms have formed. However, the recommendations for delayed infections (2–10 weeks) are not clearly defined. Here, infection healing and bone healing in early and delayed FRI is investigated in a rabbit model with a standardized DAIR procedure.

Fracture non-union can be as high as 20% in certain clinical scenarios and has a high associated socioeconomic burden. Boron has been shown to regulate the Wnt/β-catenin pathway in other bodily processes. However, this pathway is also critical for bone healing. Here we aim to demonstrate that the local delivery of boric acid can accelerate bone healing, as well as to elucidate how boric acid, via the regulationtheWnt/β-catenin pathway, impacts theosteogenic response of bone-derived osteoclasts and osteoblasts during each phase of bone repair.

Bilateral femoral cortical defects were created in 32 skeletally mature C57 mice. On the experimental side, boric acid (8mg/kg concentration) was injected locally at the defect site whereas on the control side, saline was used. Mice were euthanized at 7, 14, and 28 days. MicroCT was used to quantify bone regeneration at the defect. Histological staining for ALP and TRAP was used to quantify osteoblast and osteoclast activity respectively. Immunohistochemical antibodies, β-catenin and CD34 were used to quantify active β-catenin levels and angiogenesis respectively. Sclerostin and GSK3β were also quantified and are both inhibitors of the wnt signaling pathway via degradation and inactivation of β-catenin.

The boron group exhibited higher bone volume and trabecular thickness at the defect site by 28 days on microCT. ALP activity was significantly higher in boron group at 7 days whereas boron had no effect on TRAP activity. Additionally, CD34 staining revealed increased angiogenesis at 14 days in boron treated groups. β-catenin activity on immunohistochemistry was significantly higher in the boron group at 7 days, GSK3β was significantly higher in the boron group at 14 days and Sclerostin was significantly higher in the boron group at 28 days.

Boron appears to increase osteoblast activity at the earlier phases of healing. The corresponding early increase in β-catenin along with ALP likely supports that boron increases osteoblast activity via the wnt/β-catenin pathway. Increased angiogenesis at 14 days could be a separate mechanism increasing bone formation independent of wnt/β-catenin activation. Neither GSK3β or Sclerostin levels correlated with β-catenin activity therefore boron likely increases β-catenin through a mechanism independent of both GSK3β and Sclerostin. The addition of this inexpensive and widely available ion could potentially become a non-invasive, cost-effective treatment modality to augment fracture healing and decrease non-union rates in high risk patients.

Tendinopathy is a tendon pathology often resulting from a failed healing response to tendon injury. Activated protein C (APC) is a natural anti-coagulant with anti-inflammatory and wound healing promoting functions, which are mainly mediated by its receptors, endothelial protein C receptor (EPCR) and protease activated receptors (PARs). This study aimed to determine whether APC stimulates tenocyte healing and if so, to assess the involvement of the receptors.

Mouse-tail tenocytes were isolated from 3-week-old wild type (WT), PAR- 1 knockout (KO) and PAR-2 KO mice. The expression of EPCR, PAR-1 and −2 and the effect of APC on tenocytes tendon healing and the underlying mechanisms were investigated by Reverse transcription real time PCR, western blot, 3- (4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay, zymography, and scratch wound healing/ migration assay.

When compared to WT cells, PAR-1 KO tenocytes showed increased cell proliferation (3.3-fold, p<0.0001), migration (2.7-fold, p<0.0001) and wound healing (3-fold, p<0.0001), whereas PAR-2 KO cells displayed decreased cell proliferation (0.6-fold, p<0.05) and no change in cell migration or wound healing. APC at 1 μg/ml stimulated WT and PAR-1 KO tenocyte proliferation (~1.3, respectively, p<0.05) and wound healing (~1.3-fold, respectively, p<0.05), and additionally promoted PAR1-KO cell migration (1.4-fold, p<0.0001). APC only increased the migration (2-fold, p<0.05) of PAR-2 KO tenocytes. The activation of AKT, extracellular signal-regulated kinase (ERK)-2, and glycogen synthase kinase (GSK)-β3, the intracellular molecules that are associated with cell survival/growth, and matrix metalloproteinase (MMP)-2 that is related to cell migration and wound healing, were increased in all three cell lines in response to APC treatment.

These findings show that PAR-1 and PAR-2 act differentially in tenocyte proliferation/migration/wound healing. APC likely promotes tenocyte proliferation/ wound healing via PAR-2, not PAR-1.

Introduction

Treatment of non-union in open tibial fractures Gustilo-Anderson(GA)-3A/3B fractures remains a challenging problem. Most of these can be dealt using treatment methods that requires excision of the non-union followed by bone grafting, masquelet technique, or acute shortening. Circular fixators with closed distraction or bone transport also remains a useful option. However, sometimes due to patient specific factors these cannot be used. Recently antibiotic loaded bone substitutes have been increasingly used for repairing infected non-unions. They provide local antibiotic delivery, fill dead space, and act as a bone conductive implant, which is resorted at the end of a few months. We aimed to assess the outcome of percutaneous injection of bone substitute while treating non-union of complex open tibial fractures.

Fractures of the ankle are common, and they mostly affect young adults. Wound complications are not uncommon following the fixation of these fractures. This study evaluated the impact of HIV on wound healing after plate osteosynthesis in patients with closed ankle fractures.

This is an observational retrospective study of patients operated on at a tertiary level hospital. We reviewed hospital records for patients above 18 years of age who presented with wound breakdown following ankle open reduction and internal fixation. The patients’ hospital records were retrieved to identify all the patients treated for closed ankle fractures and those who developed wound breakdown. Patients with Pilon fractures were excluded. The National Health Laboratory System (NHLS) database was accessed to retrieve the CD4 count, viral load, haematology study results, and biochemistry results of these patients at the time of surgery and subsequent follow-up. The x-rays were retrieved from the electronic picture archiving system (PACS) and were assessed for fracture union at a minimum of 3 months follow-up.

We reviewed the medical records of 172 patients with closed ankle fractures treated from 2018 to 2022. Thirty-one (18.0%) developed wound breakdown after surgery, and they were all tested for HIV. Most of the patients were male (58.0%), and the average age of the cohort was 43.7 years (range: 21 years to 84 years). Ten of these patients (32.2%) were confirmed HIV positive, with CD4 counts ranging from 155 to 781. Viral load levels were lower than detectable in 40% of these patients. All patients progressed to fracture union at a minimum of 3 months follow-up.

We observed no difference between HIV-positive and HIV-negative patients in terms of wound breakdown and bone healing post-plate osteosynthesis for closed ankle fractures.

Mesenchymal stem cells (MSCs) are capable of forming bone, cartilage and other mesenchymal tissues but are also important modulators of innate and adaptive immune responses. We have capitalized on these important functions to mitigate adverse responses when bone is exposed to pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), or prolonged pro-inflammatory cytokines. Our goal was to optimize osteogenesis and mitigate persistent undesired inflammation by: 1. preconditioning MSCs by short term exposure to lipopolysaccharide (LPS) and Tumor Necrosis Factor alpha (TNF-α), 2. genetic modification of MSCs to overexpress Interleukin 4 (IL-4) either constitutively, or as NFκB-responsive IL-4 over-expression cells, and 3. training the MSCs (innate immune memory) by repeated stimulation with LPS.

In the first experiment, bone marrow MSCs and macrophages were isolated from femurs and tibias of C57BL/6 mice. MSCs (1×104 cells) were seeded in 24-well transwell plates in the bottom chamber with MSC growth medium. MSCs were treated with 20 ng/ml TNF-α and 1–20 μg/ml LPS for three days. Primary macrophages (2 × 103 cells) were seeded to the insert of a separate transwell plate and polarized into the M1 phenotype. At day four, MSCs and macrophages were washed and the inserts with M1 macrophages were moved to the plates containing preconditioned MSCs at the bottom of the well. Co-culture was carried out in MSC growth medium for 24h.

In the second experiment, bone marrow derived macrophages and MSCs were isolated from femora and tibiae of Balb/c male mice. 5×104 macrophages and 1×104 MSCs were seeded in the bottom well of the 24-well transwell plate. The upper chambers were seeded with unmodified MSCs, MSCs preconditioned with 20 ng/ml TNF-α and 20 mg/ml LPS for 3 days, NFκB-IL4 secreting MSCs (all 5×104 cells), or controls without MSCs. Co-culture was carried out in mixed osteogenic-macrophage media with clinically relevant polyethylene or titanium alloy particles.

In the third experiment, bone marrow MSCs and macrophages were collected from femurs and tibias of C57BL/6 male mice. The MSCs were stimulated by LPS, washed out for five days, and re-stimulated by LPS in co-culture with macrophages.

First, preconditioned MSCs enhanced anti-inflammatory M2 macrophage (Arginase 1 and CD206) expression, decreased pro-inflammatory M1 macrophage (TNF-α/IL-1Ra ratio) expression, and increased osteogenic markers (alkaline phosphatase expression and matrix mineralization) in co-culture. Second, NFκB-IL4 secreting MSCs decreased pro-inflammatory M1 (TNF-α), increased anti-inflammatory M2 (Arg1, IL-1ra) expression, and enhanced the expression of osteogenic factors Runx2 and alkaline phosphatase, in the presence of particles, compared to other groups.

Third, LPS-trained MSCs increased anti-inflammatory (Arginase1 and CD206), and decreased the proinflammatory (TNF-α, IL1b, iNOS, and IL6) marker expression in MSC/macrophage co-culture.

Transforming MSCs via the techniques of preconditioning, genetic modification, or training (innate immune memory) can modulate/convert a potentially injurious microenvironment to an anti-inflammatory pro-reconstructive milieu. These effects are highly relevant for bone healing in the presence of adverse stimuli. These concepts using transformed MSCs could also be extended to other organ systems subjected to potentially damaging agents.

The aim was to analyze whether non-steroidal anti-inflammatory drugs (NSAIDs) have an adverse effect on bone healing by evaluating all available human randomized controlled trials (RCTs) on this subject.

A systematic search of electronic databases (PubMed, MEDLINE, and Cross-References) was performed to identify RCTs comparing the occurrence of nonunion in patients who received NSAIDs to the control group. Risk of bias of the studies was assessed. Nonunion was the main outcome evaluated, however, regression analysis was used to estimate the relative risk comparing duration and type of NSAIDs.

Six RCTs (609 patients) were included. The risk of nonunion was higher in the patients given NSAIDs after the fracture (P-value= 0.0009, relative risk [RR] = 2.9, 95% confidence interval [CI] = 1.6 to 6.3). However, once the studies have been categorized to the duration of NSAIDs, those who received short period of NSAIDs (4 weeks) (P-value = 0.0002, RR = 4.1, CI = 2.1 to 8). Also, indomethacin agent has associated with high nonunion (P-value = 0.0001, RR = 3.9, CI = 2.3 to 13.9) compared to other NSAIDs which did not show a nonunion risk (P-value = 0.24, RR = 2.3, CI = 0.6 to 8.9).

Using NSAIDs for long period (> 4 weeks) after fracture is significantly associated with nonunion especially with indomethacin agent. However, short period of NSAIDs (< 2 weeks) did not show the adverse effects of nonunion. Overall, further studies are required to support our conclusion.

Previously, we reported impaired biomechanical bone properties and inferior bone matrix quality in tachykinin1 (Tac1)-deficient mice lacking the sensory neuropeptide substance P (SP). Additionally, fracture callus development is affected by the absence of SP indicating a critical effect of sensory nerve fibers on bone health and regeneration. For α-calcitonin gene-related peptide (α-CGRP)-deficient mice, a profound distortion of bone microarchitecture has also been described. We hypothesize that SP and α-CGRP modulate inflammatory as well as pain-related processes and positively affect bone regeneration during impaired fracture healing under osteoporotic conditions. Therefore, this study investigates the effects of SP and α-CGRP on fracture healing and fracture-related pain processes under conditions of experimental osteoporosis using SP- and α-CGRP-deficient mice and WT controls.

We ovariectomized female WT, Tac1−/− and α-CGRP−/− mice (age 10 weeks, all strains on C57Bl/6J background) and set intramedullary fixed femoral fractures in the left femora 28 days later. We analyzed pain threshold (Dynamic Plantar Aesthesiometer Test) and locomotion (recorded at day and night, each for 1 hour, EthoVision®XT, Noldus) at 5, 9, 13, 16 and 21 days after fracture. At each time point, fractured femora were prepared for histochemical analysis of callus tissue composition (alcian blue/sirius red staining).

Pain threshold is significantly higher in Tac1−/− mice 13 days after fracture and tends to be higher after 21 days compared to WT controls. In contrast, touch sensibility was similar in α-CGRP−/− mice and WT controls but compared to Tac1−/− mice pain threshold was significantly lower in α-CGRP−/− mice 13 and 16 days and tends to be lower 21 days after fracture. Locomotion of Tac1−/− mice during daylight was by trend higher 9 days after fracture and significantly higher 16 days after fracture whereas nightly locomotion is reduced compared to WT mice. Analysis of locomotion during daylight or night revealed no differences between α-CGRP−/− and WT mice. During early fracture healing phase, 5 and 9 days after fracture, transition of mesenchymal to cartilaginous callus tissue tends to be faster in Tac1−/− mice compared to WT controls whereas no difference was observed during late stage of fracture healing, 13, 16 and 21 days after fracture. In contrast, callus tissue maturation seems to be similar in α-CGRP−/− and WT mice.

Our data indicate different effects of SP and α-CGRP on fracture healing under conditions of experimental osteoporosis as a model for impaired bone tissue. Lack of α-CGRP seems to have no effects, but loss of SP affects locomotion throughout osteoporotic fracture healing and fracture-related pain processes during late phases of osteoporotic fracture healing. This indicates a modified role of SP during fracture healing under impaired versus healthy conditions, where SP changed early fracture-related pain processes and had no influence on callus tissue composition.

Patellar fractures account for approximately 1% of all fractures. Open reduction and internal fixation is recommended to restore extensor continuity and articular congruity. However, complications such as nonunion and symptomatic hardware, still exist. Furthermore, there is a risk of re-fracturing of the healed bone during the removal of the implants. Magnesium (Mg), a biodegradable metal, has elastic moduli and compressive yield strength that are comparable to those of natural bone. Our previous study showed that released Mg ions enhanced fracture healing. However, Mg-based implants degrade rapidly after implantation and lead to insufficient mechanical strength to support the fracture. Microarc oxidation (MAO) is a metal surface coating that reduces corrosion. We hypothesized that Mg pins, with or without MAO, would enhance fracture healing radiologically, mechanically, and histologically, while MAO would decrease degradation of Mg pins.

Patellar fracture was performed on forty-eight 18-week-old female New Zealand White rabbits according to established protocol. Briefly, the patella is osteotomized transversely and a tunnel (1.1mm) was drilled longitudinally through the two bone fragments. A pin (1 mm, stainless steel, Mg, or MAO-Mg) was inserted into the tunnel. The reduced construct was stabilized with a figure-of-eight band wire (⊘ 0.6 mm stainless steel wire). Cast immobilization was applied for 6 weeks. The rabbits were euthanized at week 8 and 12 post-operation. Microarchitecture and mechanical properties of the repaired patella were analyzed with microCT and tensile testing respectively. Histological sections of the repaired patella were stained. To evaluate the effect of the MAO treatment on degradation rate of Mg pin, the volume of the Mg pins in the patella was measured with microCT.

At week 8, both Mg and Mg-MAO showed higher ratio of bone volume to tissue volume (BV/TV) than the control while there was no significant different between Mg and Mg-MAO. At week 12, Control, Mg, and Mg-MAO groups showed enlarged patella when compared to the normal patella. Tissue volume (TV) and bone volume (BV) of the patella in Mg and Mg-MAO were larger than those in the Control group. However, the Control had higher ratio of bone volume to tissue volume (BV/TV), TV density, and BV density than Mg and Mg-MAO. Tensile testing showed that the mechanical properties of the repaired patella (failure load, stiffness, ultimate strength, and energy-to-failure) of Mg and Mg-MAO were higher than that of the control at both week 8 and week 12. Histological analysis showed that there was significant new bone formation in the Mg and Mg-MAO group compared with the Control group at week 8 and 12. The degradation rate of the MAO-coated Mg pins was significantly slower than those without MAO at week 8 but no significant difference was detected at week 12.

Mechanical, microarchitectural, and histological assessments showed that Mg pins, with or without MAO, enhanced fracture healing of the repaired patella compared to the Control. MAO treatment enhanced the corrosion resistance of the Mg pins at the early time point.

It has been previously shown that Low-Magnitude High-Frequency Vibration (LMHFV) is able to enhance ovariectomy-induced osteoporotic fracture healing in rats. Fracture healing begins with the inflammatory stage, and all subsequent stages are regulated by the infiltration of immune cells such as macrophages and the release of inflammatory cytokines including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-10 (IL-10). Therefore, the aim of this study was to investigate the effect of LMFHV treatment on the inflammatory response in osteoporotic fracture healing.

In this study, ovariectomy-induced osteoporotic and sham-operated closed-femoral fracture SD-rats were randomized into three groups: sham control (SHAM), ovariectomized control (OVX-C) or ovariectomized vibration (OVX-V) (n=36, n=6 per group per time point). LMHFV (35Hz, 0.3g) was given 20 min/day and 5 days/week to OVX-V group. SHAM operation and ovariectomy were performed at 6-month and closed femoral fracture was performed at 9-month. Callus morphometry was determined by callus width from weekly radiography. Local expressions of inducible nitric oxide synthase (iNOS) (macrophage M1 marker), CD206 (macrophage M2 marker), TNF-α, IL-6 and IL-10 were detected by immunohistochemistry and quantified by colour threshold in ImageJ, assessed at weeks 1 and 2 post-fracture. Significant difference between groups was considered at p≤0.05 by one-way ANOVA.

Callus formation was higher in OVX-V than that of OVX-C as shown by callus width at weeks 1 and 2 (p=0.054 and 0.028, respectively). Immunohistochemistry results showed that CD206 positive signal and the M2/M1 ratio which indicates the progression of macrophage polarization were significantly higher in OVX-V rats (p=0.053 and 0.049, respectively) when compared to OVX-C at week 1. Area fraction of TNF-α positive signal was significantly higher in SHAM and OVX-V rats at week 1 (p=0.01 and 0.033, respectively). IL-6 signal was also significantly higher in SHAM and OVX-V groups at week 1 (p=0.004 and 0.029, respectively). IL-10 expression was significantly lower in SHAM and OVX-V groups at week 1 (p=0.013 and 0.05, respectively).

Here we have shown that LMHFV treatment promoted the shift from pro-inflammatory stage towards anti-inflammatory stage earlier. It has been reported that the polarization of pro-inflammatory macrophages M1 to anti-inflammatory macrophages M2 was indicative of the endochondral ossification process in the long bone fracture model. Besides, we found that LMHFV treatment enhanced pro-inflammatory markers of TNF-α and IL-6 and suppressed anti-inflammatory marker of IL-10 at week 1, showing that inflammatory response was enhanced at week 1 post-fracture. These inflammatory cytokines involved in fracture healing were shown to coordinate different fracture healing processes such as mesenchymal stem cell recruitment and angiogenesis. Our previous study has demonstrated that ovariectomized rats exhibit lower levels of inflammatory response after fracture creation. Therefore, we report that LMHFV treatment can modulate macrophage polarization from M1 to M2 at an earlier time-point and partly restore the impaired inflammatory response in OVX bones at the early stage of fracture healing that may lead to accelerated healing of osteoporotic fracture as shown by promoted callus formation.