Quantifying Bone and Skin Movement in the Residual Limb-Socket Interface of Individuals With Transtibial Limb Loss Using Dynamic Stereo X-Ray: Protocol for a Lower Limb Loss Cadaver and Clinical Study.

BACKGROUND: Relative motion between the residual limb and socket in individuals with transtibial limb loss can lead to substantial consequences that limit mobility. Although assessments of the relative motion between the residual limb and socket have been performed, there remains a substantial gap in understanding the complex mechanics of the residual limb-socket interface during dynamic activities that limits the ability to improve socket design. However, dynamic stereo x-ray (DSX) is an advanced imaging technology that can quantify 3D bone movement and skin deformation inside a socket during dynamic activities. OBJECTIVE: This study aims to develop analytical tools using DSX to quantify the dynamic, in vivo kinematics between the residual limb and socket and the mechanism of residual tissue deformation. METHODS: A lower limb cadaver study will first be performed to optimize the placement of an array of radiopaque beads and markers on the socket, liner, and skin to simultaneously assess dynamic tibial movement and residual tissue and liner deformation. Five cadaver limbs will be used in an iterative process to develop an optimal marker setup. Stance phase gait will be simulated during each session to induce bone movement and skin and liner deformation. The number, shape, size, and placement of each marker will be evaluated after each session to refine the marker set. Once an optimal marker setup is identified, 21 participants with transtibial limb loss will be fitted with a socket capable of being suspended via both elevated vacuum and traditional suction. Participants will undergo a 4-week acclimation period and then be tested in the DSX system to track tibial, skin, and liner motion under both suspension techniques during 3 activities: treadmill walking at a self-selected speed, at a walking speed 10% faster, and during a step-down movement. The performance of the 2 suspension techniques will be evaluated by quantifying the 3D bone movement of the residual tibia with respect to the socket and quantifying liner and skin deformation at the socket-residuum interface. RESULTS: This study was funded in October 2021. Cadaver testing began in January 2023. Enrollment began in February 2024. Data collection is expected to conclude in December 2025. The initial dissemination of results is expected in November 2026. CONCLUSIONS: The successful completion of this study will help develop analytical methods for the accurate assessment of residual limb-socket motion. The results will significantly advance the understanding of the complex biomechanical interactions between the residual limb and the socket, which can aid in evidence-based clinical practice and socket prescription guidelines. This critical foundational information can aid in the development of future socket technology that has the potential to reduce secondary comorbidities that result from complications of poor prosthesis load transmission. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/57329.

Novel approaches to correlate computerized tomography imaging of bone fracture callus to callus structural mechanics.

Estimating the mechanical properties of bone in vivo without destructive testing would be useful for research and clinical orthopedic applications. Micro-computerized tomography (µCT) imaging can provide quantitative, high-resolution 3D representations of bone morphology and is generally the basis from which bone mechanical properties are non-destructively estimated. The goal of this study was to develop metrics using qualitative and quantitative aspects of bone microarchitecture derived from µCT imaging to estimate the mechanical integrity of bone fracture calluses. Mechanical testing data (peak torque) and µCT image data from 12 rat femur fractures were collected at 4 weeks after fracture. MATLAB was used to analyze the callus µCT imaging data which were then correlated to the empirically determined peak torque of the callus. One metric correlated Z-rays, linear contiguities of voxels running parallel to the neutral axis of the femur and through the fracture callus, to peak torque. Other metrics were based on voxel linkage values (LVs), which is a novel measurement defined by the number of voxels surrounding a given voxel (ranging from 1 to 27) that are all above a specified threshold. Linkage values were utilized to segment the callus and compute healing scores (termed eRUST) based on the modified Radiographic Union Score for Tibial fractures (mRUST). Linkage values were also used to calculate linked bone areas (LBAs). All metrics positively correlated with peak torque, yielding correlations of determination (R2) of 0.863 for eRUST, 0.792 for Z-ray scoring, and 0.764 for a normalized Linked Bone Area metric. These novel metrics appear to be promising approaches for extrapolating fracture callus structural properties from bone microarchitecture using objective analytical methods and without resorting to computationally complex finite element analyses.

Effect of Vancomycin Applied to the Surgical Site on Fracture Healing in a Diabetic Rat Model.

BACKGROUND: Prophylactic vancomycin treatment decreases the prevalence of surgical site and deep infections by >70% in diabetic patients undergoing reconstructive foot and ankle surgery. Thus, determining whether clinically relevant local vancomycin doses affect diabetic fracture healing is of medical interest. We hypothesized that application of vancomycin powder to the fracture site during surgery would not affect healing outcomes, but continuous exposure of vancomycin would inhibit differentiation of osteoblast precursor cells and their osteogenic activity in vitro. METHODS: The vancomycin dose used to treat the diabetic rats was a modest increase to routine surgical site vancomycin application of 1 to 2 g for a 70-kg adult (21 mg/kg). After femur fracture in BB-Wistar type 1 diabetic rats, powdered vancomycin (25 mg/kg) was administered to the fracture site. Bone marrow and periosteal cells isolated from diabetic bones were cultured and treated with increasing levels of vancomycin (0, 5, 50, 500, or 5000 µg/mL). RESULTS: Radiographic scoring, micro-computed tomography (µCT) analysis, and torsion mechanical testing failed to identify any statistical difference between the vancomycin-treated and the untreated fractured femurs 6 weeks postfracture. Low to moderate levels of vancomycin treatment (5 and 50 µg/mL) did not impair cell viability, osteoblast differentiation, or calcium deposition in either the periosteum or bone marrow-derived cell cultures. In contrast, high doses of vancomycin (5000 µg/mL) did impair viability, differentiation, and calcium deposition. CLINICAL RELEVANCE: In this diabetic rodent fracture model, vancomycin powder application at clinically relevant doses did not affect fracture healing or osteogenesis.

Local zinc treatment enhances fracture callus properties in diabetic rats.

The effects of locally applied zinc chloride (ZnCl2 ) on early and late-stage parameters of fracture healing were evaluated in a diabetic rat model. Type 1 Diabetes has been shown to negatively impact mechanical parameters of bone as well as biologic markers associated with bone healing. Zinc treatments have been shown to reverse those outcomes in tests of nondiabetic and diabetic animals. This study is the first to assess the efficacy of a noncarrier mediated ZnCl2 on bony healing in diabetic animals. This is a promising basic science approach which may lead to benefits for diabetic patients in the future. Treatment and healing were assessed through quantification of callus zinc, radiographic scoring, microcomputed tomography (µCT), histomorphometry, and mechanical testing. Local ZnCl2 treatment increased callus zinc levels at 1 and 3 days after fracture (p ≤ 0.025). Femur fractures treated with ZnCl2 showed increased mechanical properties after 4 and 6 weeks of healing. Histomorphometry of the ZnCl2 -treated fractures found increased callus cartilage area at Day 7 (p = 0.033) and increased callus bone area at Day 10 (p = 0.038). In contrast, callus cartilage area was decreased (p < 0.01) after 14 days in the ZnCl2 -treated rats. µCT analysis showed increased bone volume in the fracture callus of ZnCl2 -treated rats at 6 weeks (p = 0.0012) with an associated increase in the proportion of µCT voxel axial projections (Z-rays) spanning the fracture site. The results suggest that local ZnCl2 administration improves callus chondrogenesis leading to greater callus bone formation and improved fracture healing in diabetic rats.

Improved osteogenesis in rat femur segmental defects treated with human allograft and zinc adjuvants.

Bone allograft is widely used to treat large bone defects or complex fractures. However, processing methods can significantly compromise allograft osteogenic activity. Adjuvants that can restore the osteogenic activity of processed allograft should improve clinical outcomes. In this study, zinc was tested as an adjuvant to increase the osteogenic activity of human allograft in a Rag2 null rat femoral defect model. Femoral defects were treated with human demineralized bone matrix (DBM) mixed with carboxy methyl cellulose containing ZnCl2 (0, 75, 150, 300 µg) or Zn stearate (347 µg). Rat femur defects treated with DBM-ZnCl2 (75 µg) and DBM-Zn stearate (347 µg) showed increased calcified tissue in the defect site compared to DBM alone. Radiograph scoring and µCT (microcomputed tomography) analysis showed an increased amount of bone formation at the defects treated with DBM-Zn stearate. Use of zinc as an adjuvant was also tested using human cancellous bone chips. The bone chips were soaked in ZnCl2 solutions before being added to defect sites. Zn adsorbed onto the chips in a time- and concentration-dependent manner. Rat femur defects treated with Zn-bound bone chips had more new bone in the defects based on µCT and histomorphometric analyses. The results indicate that zinc supplementation of human bone allograft improves allograft osteogenic activity in the rat femur defect model.

Local insulin application has a dose-dependent effect on lumbar fusion in a rabbit model.

The purpose of this study was to determine if locally applied insulin has a dose-responsive effect on posterolateral lumbar fusion. Adult male New Zealand White rabbits underwent posterolateral intertransverse spinal fusions (PLFs) at L5-L6 using suboptimal amounts of autograft. Fusion sites were treated with collagen sponge soaked in saline (control, n = 11), or with insulin at low (5 or 10 units, n = 13), mid (20 units, n = 11), and high (40 units, n = 11) doses. Rabbits were euthanized at 6 weeks. The L5-L6 spine segment underwent manual palpation and radiographic evaluation performed by two fellowship trained spine surgeons blinded to treatment. Differences between groups were evaluated by analysis of variance on ranks followed by post-hoc Dunn's tests. Forty-three rabbits were euthanized at the planned 6 weeks endpoint, while three died or were euthanized prior to the endpoint. Radiographic evaluation found bilateral solid fusion in 10%, 31%, 60%, and 60% of the rabbits from the control and low, mid, and high-dose insulin-treated groups, respectively (p < 0.05). As per manual palpation, 7 of 10 rabbits in the mid-dose insulin group were fused as compared to 1 of 10 rabbits in the control group (p < 0.05). This study demonstrates that insulin enhanced the effectiveness of autograft to increase fusion success in the rabbit PLF model. The study indicates that insulin or insulin-mimetic compounds can be used to promote bone regeneration.

Naproxen treatment inhibits articular cartilage loss in a rat model of osteoarthritis.

The effects of naproxen, a nonsteroidal anti-inflammatory drug (NSAID), on articular cartilage degeneration in female Sprague-Dawley rats was examined. Osteoarthritis (OA) was induced by destabilization of the medial meniscus (DMM) in each knee. Rats were treated with acetaminophen (60 mg/kg), naproxen (8 mg/kg), or 1% carboxymethylcellulose (placebo) by oral gavage twice daily for 3 weeks, beginning 2 weeks after surgery. OA severity was assessed by histological Osteoarthritis Research Society International (OARSI) scoring and by measuring proximal tibia cartilage depth using contrast enhanced µCT (n = 6 per group) in specimens collected at 2, 5, and 7 weeks after surgery as well as on pristine knees. Medial cartilage OARSI scores from the DMM knees of naproxen-treated rats were statistically lower (i.e., better) than the medial cartilage OARSI scores from the DMM knees of placebo-treated rats at 5-weeks (8.7 ± 3.6 vs. 13.2 ± 2.4, p = 0.025) and 7-weeks (9.5 ± 1.2 vs. 12.5 ± 2.5, p = 0.024) after surgery. At 5 weeks after DMM surgery, medial articular cartilage depth in the proximal tibia specimens was significantly greater in the naproxen (1.78 ± 0.26 mm, p = 0.005) and acetaminophen (1.94 ± 0.12 mm, p < 0.001) treated rats as compared with placebo-treated rats (1.34 ± 0.24 mm). However, at 7 weeks (2 weeks after drug withdrawal), medial articular cartilage depth for acetaminophen-treated rats (1.36 ± 0.29 mm) was significantly reduced compared with specimens from the naproxen-treated rats (1.88 ± 0.14 mm; p = 0.004). The results indicate that naproxen treatment reduced articular cartilage degradation in the rat DMM model during and after naproxen treatment.

Zinc as a Therapeutic Agent in Bone Regeneration.

Zinc is an essential mineral that is required for normal skeletal growth and bone homeostasis. Furthermore, zinc appears to be able to promote bone regeneration. However, the cellular and molecular pathways through which zinc promotes bone growth, homeostasis, and regeneration are poorly understood. Zinc can positively affect chondrocyte and osteoblast functions, while inhibiting osteoclast activity, consistent with a beneficial role for zinc in bone homeostasis and regeneration. Based on the effects of zinc on skeletal cell populations and the role of zinc in skeletal growth, therapeutic approaches using zinc to improve bone regeneration are being developed. This review focuses on the role of zinc in bone growth, homeostasis, and regeneration while providing an overview of the existing studies that use zinc as a bone regeneration therapeutic.

Deletion of FOXO1 in chondrocytes rescues the effect of diabetes on mechanical strength in fracture healing.

Diabetes increases the risk of fracture, impairs fracture healing and causes rapid loss of the fracture callus cartilage, which was linked to increased FOXO1 expression in chondrocytes. We recently demonstrated that deletion of FOXO1 in chondrocytes blocked the premature removal of cartilage associated with endochondral bone formation during fracture healing. However, the ultimate impact of this deletion on mechanical strength was not investigated and remains unknown. Closed fractures were induced in Col2α1Cre+.FOXO1L/L mice with lineage specific deletion of FOXO1 in chondrocytes compared to littermate controls. Type 1 diabetes was induced by multiple low dose streptozotocin treatment. Thirty-five days after fracture micro CT analysis showed that diabetes significantly reduced callus volume and bone volume (P < 0.05), both which were reversed by FOXO1 deletion in chondrocytes. Diabetes significantly reduced mechanical strength measured by maximum torque, stiffness, modulus of rigidity and toughness and FOXO1 deletion in diabetic mice rescued each parameter (P < 0.05). Diabetes also reduced both bone volume and mechanical strength in non-fractured femurs. However, FOXO1 deletion did not affect bone volume or strength in non-fractured bone. These results point to the important effect that diabetes has on chondrocytes and show for the first time that the premature removal of cartilage induced by FOXO1 in chondrocytes has a significant impact on the mechanical strength of the healing bone.

Chondrocytes Promote Vascularization in Fracture Healing Through a FOXO1-Dependent Mechanism.

Chondrocytes play an essential role in fracture healing by producing cartilage, which forms an anlage for endochondral ossification that stabilizes the healing fracture callus. More recently it has been appreciated that chondrocytes have the capacity to produce factors that may affect the healing process. We examined the role of chondrocytes in angiogenesis during fracture healing and the role of the transcription factor forkhead box-O 1 (FOXO1), which upregulates wound healing in soft tissue. Closed fractures were induced in experimental mice with lineage-specific FOXO1 deletion by Cre recombinase under the control of a collagen-2α1 promoter element (Col2α1Cre+ FOXO1L/L ) and Cre recombinase negative control littermates containing flanking loxP sites (Col2α1Cre- FOXO1L/L ). Experimental mice had significantly reduced CD31+ new vessel formation. Deletion of FOXO1 in chondrocytes in vivo suppressed the expression of vascular endothelial growth factor-A (VEGFA) at both the protein and mRNA levels. Overexpression of FOXO1 in chondrocytes in vitro increased VEGFA mRNA levels and VEGFA transcriptional activity whereas silencing FOXO1 reduced it. Moreover, FOXO1 interacted directly with the VEGFA promoter and a deacetylated FOXO1 mutant enhanced VEGFA expression whereas an acetylated FOXO1 mutant did not. Lastly, FOXO1 knockdown by siRNA significantly reduced the capacity of chondrocytes to stimulate microvascular endothelial cell tube formation in vitro. The results indicate that chondrocytes play a key role in angiogenesis which is FOXO1 dependent and that FOXO1 in chondrocytes regulates a potent angiogenic factor, VEGFA. These studies provide new insight into fracture healing given the important role of vessel formation in the fracture repair process. © 2018 American Society for Bone and Mineral Research.

FOXO1 Deletion Reverses the Effect of Diabetic-Induced Impaired Fracture Healing.

Type 1 diabetes impairs fracture healing. We tested the hypothesis that diabetes affects chondrocytes to impair fracture healing through a mechanism that involves the transcription factor FOXO1. Type 1 diabetes was induced by streptozotocin in mice with FOXO1 deletion in chondrocytes (Col2α1Cre+FOXO1L/L) or littermate controls (Col2α1Cre-FOXO1L/L) and closed femoral fractures induced. Diabetic mice had 77% less cartilage and 30% less bone than normoglycemics evaluated histologically and by micro-computed tomography. Both were reversed with lineage-specific FOXO1 ablation. Diabetic mice had a threefold increase in osteoclasts and a two- to threefold increase in RANKL mRNA or RANKL-expressing chondrocytes compared with normoglycemics. Both parameters were rescued by FOXO1 ablation in chondrocytes. Conditions present in diabetes, high glucose (HG), and increased advanced glycation end products (AGEs) stimulated FOXO1 association with the RANKL promoter in vitro, and overexpression of FOXO1 increased RANKL promoter activity in luciferase reporter assays. HG and AGE stimulated FOXO1 nuclear localization, which was reversed by insulin and inhibitors of TLR4, histone deacetylase, nitric oxide, and reactive oxygen species. The results indicate that chondrocytes play a prominent role in diabetes-impaired fracture healing and that high levels of glucose, AGEs, and tumor necrosis factor-α, which are elevated by diabetes, alter RANKL expression in chondrocytes via FOXO1.

Osteoclast depletion with clodronate liposomes delays fracture healing in mice.

Osteoclasts are abundant within the fracture callus and also localize at the chondro-osseous junction. However, osteoclast functions during fracture healing are not well defined. Inhibition of osteoclast formation or resorptive activity impairs callus remodeling but does not prevent callus formation. Interestingly, though anti-osteoclast therapies differentially affect resolution of callus cartilage into bone. Treatments that inhibit osteoclast formation or viability tend to impair callus cartilage resolution, while treatments that target inhibition of bone resorption generally do not affect callus cartilage resolution. Here, we tested whether depletion of osteoclasts by systemic treatment with clodronate liposomes would similarly impair callus cartilage resolution. ICR mice were treated by intraperitoneal injections of clodronate-laden liposomes or control liposomes and subjected to closed femur fracture. Femurs were resected at multiple times after fracture and analyzed by radiography, histology, and mechanical testing to determine effects on healing. Clodronate liposome treatment did not prevent callus formation. However, radiographic scoring indicated that clodronate liposome treatment impaired healing. Clodronate liposome treatment significantly reduced callus osteoclast populations and delayed resolution of callus cartilage. Consistent with continued presence of callus cartilage, torsional mechanical testing found significant decreases in callus material properties after 28 days of healing. The results support a role for osteoclasts in the resolution of callus cartilage into bone. Whether the cartilage resolution role for osteoclasts is limited to simply resorbing cartilage at the chondro-osseous junction or in promoting bone formation at the chondro-osseous junction through another mechanism, perhaps similar to the reversal process in bone remodeling, will require further experimentation. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1699-1706, 2017.

The Biology of Bone and Ligament Healing.

This review describes the normal healing process for bone, ligaments, and tendons, including primary and secondary healing as well as bone-to-bone fusion. It depicts the important mediators and cell types involved in the inflammatory, reparative, and remodeling stages of each healing process. It also describes the main challenges for clinicians when trying to repair bone, ligaments, and tendons with a specific emphasis on Charcot neuropathy, fifth metatarsal fractures, arthrodesis, and tendon sheath and adhesions. Current treatment options and research areas are also reviewed.

Effect on Clinical Outcome and Growth Factor Synthesis With Adjunctive Use of Pulsed Electromagnetic Fields for Fifth Metatarsal Nonunion Fracture: A Double-Blind Randomized Study.

BACKGROUND: Electromagnetic bone growth stimulators have been found to biologically enhance the bone healing environment, with upregulation of numerous growth factors. The purpose of the study was to quantify the effect, in vivo, of pulsed electromagnetic fields (PEMFs) on growth factor expression and healing time in fifth metatarsal nonunions. METHODS: This was a prospective, randomized, double-blind trial of patients, cared for by 2 fellowship-trained orthopedic foot and ankle surgeons. Inclusion criteria consisted of patients between 18 and 75 years old who had been diagnosed with a fifth metatarsal delayed or nonunion, with no progressive signs of healing for a minimum of 3 months. Eight patients met inclusion criteria and were randomized to receive either an active stimulation or placebo PEMF device. Each patient then underwent an open biopsy of the fracture site and was fitted with the appropriate PEMF device. The biopsy was analyzed for messenger-ribonucleic acid (mRNA) levels using quantitative competitive reverse transcription polymerase chain reaction (QT-RT-PCR). Three weeks later, the patient underwent repeat biopsy and open reduction and internal fixation of the nonunion site. The patients were followed at 2- to 4-week intervals with serial radiographs and were graded by the number of cortices of healing. RESULTS: All fractures healed, with an average time to complete radiographic union of 14.7 weeks and 8.9 weeks for the inactive and active PEMF groups, respectively. A significant increase in placental growth factor (PIGF) level was found after active PEMF treatment (P = .043). Other factors trended higher following active PEMF including brain-derived neurotrophic factor (BDNF), bone morphogenetic protein (BMP) -7, and BMP-5. CONCLUSION: The adjunctive use of PEMF for fifth metatarsal fracture nonunions produced a significant increase in local placental growth factor. PEMF also produced trends toward higher levels of multiple other factors and faster average time to radiographic union compared to unstimulated controls. LEVEL OF EVIDENCE: Level I, prospective randomized trial.

Zinc has insulin-mimetic properties which enhance spinal fusion in a rat model.

BACKGROUND CONTEXT: Previous studies have found that insulin or insulin-like growth factor treatment can stimulate fracture healing in diabetic and normal animal models, and increase fusion rates in a rat spinal fusion model. Insulin-mimetic agents, such as zinc, have demonstrated antidiabetic effects in animal and human studies, and these agents that mimic the effects of insulin could produce the same beneficial effects on bone regeneration and spinal fusion. PURPOSE: The purpose of this study was to analyze the effects of locally applied zinc on spinal fusion in a rat model. STUDY DESIGN/SETTING: Institutional Animal Care and Use Committee-approved animal study using Sprague-Dawley rats was used as the study design. METHODS: Thirty Sprague-Dawley rats (450-500 g) underwent L4-L5 posterolateral lumbar fusion (PLF). After decortication and application of approximately 0.3 g of autograft per side, one of three pellets were added to each site: high-dose zinc calcium sulfate (ZnCaSO4), low-dose ZnCaSO4 (half of the high dose), or a control palmitic acid pellet (no Zn dose). Systemic blood glucose levels were measured 24 hours postoperatively. Rats were sacrificed after 8weeks and the PLFs analyzed qualitatively by manual palpation and radiograph review, and quantitatively by micro-computed tomography (CT) analysis of bone volume and trabecular thickness. Statistical analyses with p-values set at .05 were accomplished with analysis of variance, followed by posthoc tests for quantitative data, or Mann-Whitney rank tests for qualitative assessments. RESULTS: Compared with controls, the low-dose zinc group demonstrated a significantly higher manual palpation grade (p=.011), radiographic score (p=.045), and bone formation on micro-CT (172.9 mm(3) vs. 126.7 mm(3) for controls) (p<.01). The high-dose zinc also demonstrated a significantly higher radiographic score (p=.017) and bone formation on micro-CT (172.7 mm(3) vs. 126.7 mm(3)) (p<.01) versus controls, and was trending toward higher manual palpation scores (p=.058). CONCLUSIONS: This study demonstrates the potential benefit of a locally applied insulin-mimetic agent, such as zinc, in a rat lumbar fusion model. Previous studies have demonstrated the benefits of local insulin application in the same model, and it appears that zinc has similar effects.

Variation in lipid mediator and cytokine levels during mouse femur fracture healing.

Fracture healing is regulated by a variety of inflammatory mediators and growth factors which act over time to regenerate the injured tissue. This study used a mouse femur fracture model to quantify the temporal expression pattern of lipid mediators, cytokines, and related mRNAs during healing. Cyclooxygenase (COX-1 and COX-2) and 5-lipoxygenase (5-LO) derived lipid mediators, cytokines, and mRNA levels were quantified using mass spectrometry (LC-MS/MS), bead-based multiplex assays (xMAP), and quantitative PCR of cDNA (RTqPCR), respectively. Our analysis found that, the early inflammatory response (between 0 and 4 days after fracture) in the mouse femur fracture model coincided with elevated levels of COX-derived lipid mediators and inflammatory cytokines but with decreased levels of 5-LO-derived lipid mediators. Further, the COX-derived lipid mediators remained elevated for at least 7 days after fracture, suggesting that the COX-derived lipid mediators have additional functions during later phases of the fracture healing response. Differences were also found between mRNA levels and corresponding cytokines and lipid mediator levels, supporting a role for post-transcriptional regulation of gene expression. The temporal changes in fracture callus lipid mediator levels and inflammatory cytokines support a general positive role for inflammatory cytokines and COX-derived lipid mediators on fracture healing and a general negative role for 5-lipoxygenase derived lipid mediators during the initial stages of repair. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1883-1893, 2016.

Salicylic Acid-Based Polymers for Guided Bone Regeneration Using Bone Morphogenetic Protein-2.

Bone morphogenetic protein-2 (BMP-2) is used clinically to promote spinal fusion, treat complex tibia fractures, and to promote bone formation in craniomaxillofacial surgery. Excessive bone formation at sites where BMP-2 has been applied is an established complication and one that could be corrected by guided tissue regeneration methods. In this study, anti-inflammatory polymers containing salicylic acid [salicylic acid-based poly(anhydride-ester), SAPAE] were electrospun with polycaprolactone (PCL) to create thin flexible matrices for use as guided bone regeneration membranes. SAPAE polymers hydrolyze to release salicylic acid, which is a nonsteroidal anti-inflammatory drug. PCL was used to enhance the mechanical integrity of the matrices. Two different SAPAE-containing membranes were produced and compared: fast-degrading (FD-SAPAE) and slow-degrading (SD-SAPAE) membranes that release salicylic acid at a faster and slower rate, respectively. Rat femur defects were treated with BMP-2 and wrapped with FD-SAPAE, SD-SAPAE, or PCL membrane or were left unwrapped. The effects of different membranes on bone formation within and outside of the femur defects were measured by histomorphometry and microcomputed tomography. Bone formation within the defect was not affected by membrane wrapping at BMP-2 doses of 12 µg or more. In contrast, the FD-SAPAE membrane significantly reduced bone formation outside the defect compared with all other treatments. The rapid release of salicylic acid from the FD-SAPAE membrane suggests that localized salicylic acid treatment during the first few days of BMP-2 treatment can limit ectopic bone formation. The data support development of SAPAE polymer membranes for guided bone regeneration applications as well as barriers to excessive bone formation.

Local manganese chloride treatment accelerates fracture healing in a rat model.

This study investigated the effects of local delivery of manganese chloride (MnCl2), an insulin-mimetic compound, upon fracture healing using a rat femoral fracture model. Mechanical testing, histomorphometry, and immunohistochemistry were performed to assess early and late parameters of fracture healing. At 4 weeks post-fracture, maximum torque to failure was 70% higher (P<0.05) and maximum torsional rigidity increased 133% (P<0.05) in animals treated with 0.125 mg/kg MnCl2 compared to saline controls. Histological analysis of the fracture callus revealed percent new mineralized tissue was 17% higher (P<0.05) at day 10. Immunohistochemical analysis of the 0.125 mg/kg MnCl2 treated group, compared to saline controls, showed a 379% increase in the density of VEGF-C+ cells. In addition, compared to saline controls, the 0.125 mg/kg MnCl2 treated group showed a 233% and 150% increase in blood vessel density in the subperiosteal region at day 10 post-fracture as assessed by detection of PECAM and smooth muscle α actin, respectively. The results suggest that local MnCl2 treatment accelerates fracture healing by increasing mechanical parameters via a potential mechanism of amplified early angiogenesis leading to increased osteogenesis. Therefore, local administration of MnCl2 is a potential therapeutic adjunct for fracture healing.

Method for measuring lipid mediators, proteins, and messenger RNAs from a single tissue specimen.

This article describes a new method for extracting RNA, protein, and lipid mediators from a single tissue specimen. Specifically, mouse bone fracture callus specimens were extracted into a single solution that was processed using three different procedures to measure messenger RNA (mRNA) levels by reverse transcription-quantitative polymerase chain reaction (RTqPCR), cytokines and growth factors using an xMAP method, and lipid mediators by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This method has several advantages because it decreases the number of animals necessary for experimentation, allows division of the sample from a homogeneous mixture that reduces sample variability, and uses a solution that protects the integrity of the macromolecules during storage.

Fracture healing and lipid mediators.

Lipid mediators regulate bone regeneration during fracture healing. Prostaglandins and leukotrienes are well-known lipid mediators that regulate inflammation and are synthesized from the Ω-6 fatty acid, arachidonic acid. Cyclooxygenase (COX-1 or COX-2) and 5-lipoxygenase (5-LO) catalyze the initial enzymatic steps in the synthesis of prostaglandins and leukotrienes, respectively. Inhibition or genetic ablation of COX-2 activity impairs fracture healing in animal models. Genetic ablation of COX-1 does not affect the fracture callus strength in mice, suggesting that COX-2 activity is primarily responsible for regulating fracture healing. Inhibition of cyclooxygenase activity with nonsteroidal anti-inflammatory drugs (NSAIDs) is performed clinically to reduce heterotopic ossification, although clinical evidence that NSAID treatment impairs fracture healing remains controversial. In contrast, inhibition or genetic ablation of 5-LO activity accelerates fracture healing in animal models. Even though prostaglandins and leukotrienes regulate inflammation, loss of COX-2 or 5-LO activity appears to primarily affect chondrogenesis during fracture healing. Prostaglandin or prostaglandin analog treatment, prostaglandin-specific synthase inhibition and prostaglandin or leukotriene receptor antagonism also affect callus chondrogenesis. Unlike the Ω-6-derived lipid mediators, lipid mediators derived from Ω-3 fatty acids, such as resolvin E1 (RvE1), have anti-inflammatory activity. In vivo, RvE1 can inhibit osteoclastogenesis and limit bone resorption. Although Ω-6 and Ω-3 lipid mediators have clear-cut effects on inflammation, the role of these lipid mediators in bone regeneration is more complex, with apparent effects on callus chondrogenesis and bone remodeling.