Local BMP2 hydrogel therapy for robust bone regeneration in a porcine model of Legg-Calvé-Perthes disease.

Legg-Calvé-Perthes disease is juvenile idiopathic osteonecrosis of the femoral head (ONFH) that has no effective clinical treatment. Previously, local injection of bone morphogenetic protein-2 (BMP2) for ONFH treatment showed a heterogeneous bone repair and a high incidence of heterotopic ossification (HO) due to the BMP2 leakage. Here, we developed a BMP2-hydrogel treatment via a transphyseal bone wash and subsequential injection of BMP2-loaded hydrogel. In vitro studies showed that a hydrogel of gelatin-heparin-tyramine retained the BMP2 for four weeks. The injection of the hydrogel can efficiently prevent leakage. With the bone wash, the injected hydrogel had a broad distribution in the head. In vivo studies on pigs revealed that the BMP2-hydrogel treatment produced a homogeneous bone regeneration without HO. It preserved the subchondral contour and restored the subchondral endochondral ossification, although it increased growth plate fusions. In summary, the study demonstrated a promising BMP2-hydrogel treatment for ONFH treatment, especially for teenagers.

Local Administration of Bone Morphogenetic Protein-2 Using a Hydrogel Carrier for Robust Bone Regeneration in a Large Animal Model of Legg-Calvé-Perthes disease.

Legg-Calvé-Perthes disease is juvenile idiopathic osteonecrosis of the femoral head (ONFH) that has no effective clinical resolutions. Previously, local injection of bone morphogenetic protein-2 (BMP2) for ONFH treatment showed a heterogeneous bone repair and a high incidence of heterotopic ossification (HO) due to the BMP2 leakage. Here, we developed a BMP2-hydrogel treatment via a transphyseal bone wash and subsequential injection of BMP2-loaded hydrogel. In vivo studies showed that a hydrogel of gelatin-heparin-tyramine retained the BMP2 for four weeks. The injection of the hydrogel can efficiently prevent leakage. With the bone wash, the injected hydrogel had a broad distribution in the head. In vivo studies on pigs revealed that the BMP2-hydrogel treatment produced a homogeneous bone regeneration without HO. It preserved the subchondral contour and restored the subchondral endochondral ossification, although it increased growth plate fusions. In summary, the study demonstrated a promising BMP2-hydrogel treatment for ONFH treatment, especially for teenagers.

Minimally Invasive Necrotic Bone Washing Improves Bone Healing After Femoral Head Ischemic Osteonecrosis: An Experimental Investigation in Immature Pigs.

BACKGROUND: Ischemic osteonecrosis of the femoral head produces necrotic cell debris and inflammatory molecules in the marrow space, which elicit a chronic inflammatory repair response. The purpose of this study was to determine the effects of flushing out the necrotic cell debris and inflammatory proteins on bone repair in a piglet model of ischemic osteonecrosis. METHODS: Osteonecrosis of the femoral head of the right hindlimb was induced in 12 piglets by tying a ligature tightly around the femoral neck. One week after the surgery, 6 animals were treated with a percutaneous 3-needle bone washing procedure and non-weight-bearing (NWB) of the right hindlimb (wash group). The total saline solution wash volume was 450 mL per femoral head. Serial wash solutions were collected and analyzed. The remaining 6 animals were treated with NWB only (NWB group). At 8 weeks after the surgery, the femoral heads were assessed using radiography, micro-computed tomography (micro-CT), and histological analysis. In addition, we compared the results for these piglets with our published results for 6 piglets treated with multiple epiphyseal drilling (MED) plus NWB without bone washing (MED group). RESULTS: Necrotic cells and inflammatory proteins were present in the bone wash solution collected 1 week after ischemia induction. The protein and triglyceride concentrations decreased significantly with subsequent washing (p < 0.005). At 8 weeks after ischemia induction, the wash group had a significantly higher bone volume than the MED or NWB group (p < 0.0001). Histological bone-formation measures were also significantly increased in the wash group compared with the MED group (p = 0.002) or NWB group (p < 0.0001) while macrophage numbers were significantly decreased in the wash group. CONCLUSIONS: The percutaneous 3-needle procedure flushed out cell debris and inflammatory proteins from the necrotic femoral heads, decreased osteoclasts and macrophages, and increased bone formation following induction of ischemic osteonecrosis. CLINICAL RELEVANCE: We believe that this is the first study to investigate the concept of washing out the necrotic femoral head to improve bone healing. The minimally invasive procedure may be useful to improve the necrotic bone environment and bone repair following ischemic osteonecrosis.

Local temperature elevation as a marker of spinal implant infection in an animal model.

BACKGROUND: Temperature elevation, a classic marker of infection and local temperature elevation, might be a useful predictor of early infection. However temperature measurement around the spine is not readily accessible. The purpose of this study was to explore whether a temperature sensing implant might reproducibly detect local temperature change associated with peri-implant wound infection, in a rabbit model. METHODS: Twelve adult rabbits were implanted with a spinal screw-rod construct. Temperature probes were placed at the implantation site as well as at a separate scapular site away from the surgical site to serve as control. Animals were inoculated with S. aureus: group 1 (saline control), group 2 (low dose 1 × 102 CFU/site), group 3 (medium dose 1 × 104 CFU/site), and group 4 (high dose 1 × 106 CFU/site) and monitored for 7 days prior to euthanasia. RESULTS: The scapular control temperature and implant site temperature in the non-infected animals remained similar throughout the study period. Both the scapular control and implant site temperatures were elevated in the infected animals compared to the non-infected animals. There was a statistically significant difference in the scapular control temperature and implant site temperature in all infected animals but not in the non-infected animals. Difference in temperature elevation between implant site and control scapular site were greatest for the animals with worst clinical appearance during the post-mortem evaluation. CONCLUSIONS: This rabbit model demonstrates that local temperature measured in proximity to a spinal implant is elevated in the presence of infection with greater elevations associated with worse infections. Availability of an implantable temperature sensor may yield valuable information for the assessment and treatment of suspected spinal wound infection in the clinical setting.

Anti-Interleukin-6 Therapy Decreases Hip Synovitis and Bone Resorption and Increases Bone Formation Following Ischemic Osteonecrosis of the Femoral Head.

Legg-Calvé-Perthes disease (LCPD) is a juvenile form of ischemic femoral head osteonecrosis, which produces chronic hip synovitis, permanent femoral head deformity, and premature osteoarthritis. Currently, there is no medical therapy for LCPD. Interleukin-6 (IL-6) is significantly elevated in the synovial fluid of patients with LCPD. We hypothesize that IL-6 elevation promotes chronic hip synovitis and impairs bone healing after ischemic osteonecrosis. We set out to test if anti-IL-6 therapy using tocilizumab can decrease hip synovitis and improve bone healing in the piglet model of LCPD. Fourteen piglets were surgically induced with ischemic osteonecrosis and assigned to two groups: the no treatment group (n = 7) and the tocilizumab group (15 to 20 mg/kg, biweekly intravenous injection, n = 7). All animals were euthanized 8 weeks after the induction of osteonecrosis. Hip synovium and femoral heads were assessed for hip synovitis and bone healing using histology, micro-CT, and histomorphometry. The mean hip synovitis score and the number of synovial macrophages and vessels were significantly lower in the tocilizumab group compared with the no treatment group (p < .0001, p = .01, and p < .01, respectively). Micro-CT analysis of the femoral heads showed a significantly higher bone volume in the tocilizumab group compared with the no treatment group (p = .02). The histologic assessment revealed a significantly lower number of osteoclasts per bone surface (p < .001) in the tocilizumab group compared with the no treatment group. Moreover, fluorochrome labeling showed a significantly higher percent of mineralizing bone surface (p < .01), bone formation rate per bone surface (p < .01), and mineral apposition rate (p = .04) in the tocilizumab group. Taken together, tocilizumab therapy decreased hip synovitis and osteoclastic bone resorption and increased new bone formation after ischemic osteonecrosis. This study provides preclinical evidence that tocilizumab decreases synovitis and improves bone healing in a large animal model of LCPD. © 2020 American Society for Bone and Mineral Research (ASBMR).

Development of a novel minimally invasive technique to washout necrotic bone marrow content from epiphyseal bone: A preliminary cadaveric bone study.

INTRODUCTION: Legg-Calvé-Perthes disease is a juvenile ischemic osteonecrosis which produces extensive necrotic cell debris and release of damage associated molecular patterns (DAMPs) in the femoral head. The necrotic bone environment induces a chronic inflammatory repair response with excessive bone resorption leading to deformity and early osteoarthritis. Currently there is no minimally invasive method to clear the necrotic materials from the bone to decrease the inflammatory burden of the necrotic environment and to improve the healing process. HYPOTHESIS: We hypothesized that a novel minimally invasive two-needle saline washing technique would be effective to remove cell debris, proteins, and fat from the marrow space of porcine cadaveric humeral heads (HHs). MATERIALS AND METHODS: Twenty-two HHs were subjected to three freeze-thaw cycles to simulate osteonecrosis prior to the wash procedure which consisted of placement of two 15-gauge intraosseous needles followed by incremental saline wash. After the washout procedure, the solutions were collected for measurements of turbidity, protein concentration, and cell count. The HHs were analyzed by optical scanning and histology. RESULTS: The solution collected after each wash showed a significant decrease in the turbidity, cell count, and protein concentration (p<0.05). Histologic assessment showed significantly decreased cell debris and adipocytes in the washed group compared to the unwashed group (p<0.001). DISCUSSION/CONCLUSION: The two-needle intraosseous wash technique effectively removed cell debris and proteins from the marrow space. The technique may be used to reduce the necrotic cell debris and DAMPs present in the necrotic bone. LEVEL OF EVIDENCE: III, in vitro comparative study.

A Comparison of Transphyseal Neck-Head Tunneling and Multiple Epiphyseal Drilling on Femoral Head Healing Following Ischemic Osteonecrosis: An Experimental Investigation in Immature Pigs.

BACKGROUND: Two operative procedures are currently advocated to stimulate the necrotic femoral head healing in children with Legg-Calve-Perthes disease: transphyseal neck-head tunneling (TNHT) and multiple epiphyseal drilling (MED). The purpose of this study was to compare the bone healing and physeal function after treatment with TNHT or MED in a piglet model of ischemic osteonecrosis. METHODS: Eighteen piglets were induced with osteonecrosis by surgically placing a ligature tightly around the right femoral neck. One week later, the piglets were assigned to 1 of 3 treatment groups (n=6/group): (1) local nonweight bearing only (NWB), (2) TNHT plus NWB, or (3) MED plus NWB. The unoperated left femoral heads were used as normal controls. The animals were euthanized at 8 weeks after osteonecrosis induction. Histologic, histomorphometric, radiographic, microcomputed tomography (CT), and calcein-labeling assessments were performed. Statistical analysis included a 1-way ANOVA. RESULTS: Micro-CT analyses showed higher femoral head bone volume in the MED group compared with the TNHT and the NWB groups (P<0.01). The MED group had a higher mean trabecular number (P<0.001) and new bone formation (P=0.001) based on calcein-labeling parameters compared with the TNHT and the NWB groups. In addition, the osteoclast number per bone surface was lower in the MED group compared with the NWB group (P=0.001). Histologic and micro-CT assessments of the proximal femoral physis revealed a larger physeal disruption at the site of physeal drilling in the TNHT group compared with the MED group. However, no significant differences in physeal elongation (P=0.61) and femoral neck length (P=0.31) were observed between the treatment groups. CONCLUSIONS: MED produced a higher bone volume and stimulated greater bone formation than the TNHT or the NWB alone. Both procedures did not produce a significant physeal growth disturbance during the study period. CLINICAL RELEVANCE: This preclinical study provides evidence that MED produces more favorable bone healing than the TNHT in a large animal model of Legg-Calve-Perthes disease.

Quantitative susceptibility mapping detects neovascularization of the epiphyseal cartilage after ischemic injury in a piglet model of legg-calvé-perthes disease.

BACKGROUND: Legg-Calvé-Perthes disease (LCPD) is a childhood hip disorder thought to be caused by disruption of blood supply to the developing femoral head. There is potential for imaging to help assess revascularization of the femoral head. PURPOSE: To investigate whether quantitative susceptibility mapping (QSM) can detect neovascularization in the epiphyseal cartilage following ischemic injury to the developing femoral head. STUDY TYPE: Prospective. ANIMAL MODEL: Right femoral head ischemia was surgically induced in 6-week-old male piglets. The animals were sacrificed 48 hours (n = 3) or 4 weeks (n = 7) following surgery, and the operated and contralateral control femoral heads were harvested for ex vivo MRI. FIELD STRENGTH/SEQUENCE: Preclinical 9.4T MRI to acquire susceptibility-weighted 3D gradient echo (GRE) images with 0.1 mm isotropic spatial resolution. ASSESSMENT: The 3D GRE images were used to manually segment the cartilage overlying the femoral head and were subsequently postprocessed using QSM. Vessel volume, cartilage volume, and vessel density were measured and compared between operated and control femoral heads at each timepoint. Maximum intensity projections of the QSM images were subjectively assessed to identity differences in cartilage canal appearance, location, and density. STATISTICAL TESTS: Paired t-tests with Bonferroni correction were used (P < 0.008 considered significant). RESULTS: Increased vascularity of the epiphyseal cartilage following ischemic injury was clearly identified using QSM. No changes were detected 48 hours after surgery. Vessel volume, cartilage volume, and vessel density were all increased in the operated vs. control femoral heads 4 weeks after surgery (P = 0.001, 0.002, and 0.001, respectively). Qualitatively, the increase in vessel density at 4 weeks was due to the formation of new vessels that were organized in a brush-like orientation in the epiphyseal cartilage, consistent with the histological appearance of neovascularization. DATA CONCLUSION: QSM can detect neovascularization in the epiphyseal cartilage following ischemic injury to the femoral head. LEVEL OF EVIDENCE: 1 Technical Efficacy: Stage 1 J. Magn. Reson. Imaging 2019;50:106-113.

Interleukin-6 deletion stimulates revascularization and new bone formation following ischemic osteonecrosis in a murine model.

Legg-Calvé-Perthes disease (LCPD) is a childhood form of ischemic osteonecrosis of the femoral head which can produce a permanent femoral head deformity and early osteoarthritis. The femoral head deformity results from increased bone resorption and decreased bone formation during repair and remodeling of the necrotic femoral head. A recent study showed that a pro-inflammatory cytokine, interleukin-6 (IL-6), is significantly elevated in the synovial fluid of patients with LCPD. We hypothesized that IL-6 elevation decreases bone formation during the repair process following ischemic osteonecrosis and that IL-6 depletion will increase new bone formation. To test this hypothesis, we surgically induced ischemic osteonecrosis in the wild-type (n = 29) and IL-6 knockout (KO) mice (n = 25). The animals were assessed at 48 h, 2 weeks and 4 weeks following the induction of ischemic osteonecrosis using histologic, histomorphometric and micro-CT methods. IL-6 immunohistochemistry showed high expression of IL-6 in the osteonecrotic side of the wild-type mice at 48 h and 4 weeks following ischemic osteonecrosis, but not in the IL-6 KO mice. We also confirmed an undetectable level of IL-6 expression in the primary osteoblasts of the IL-6 KO mice compared to the readily detectable level in the wild-type mice. Furthermore, we confirmed that IL-6 deletion did not affect the extent of bone necrosis in the IL-6 KO mice compared to the wild-type mice by performing histologic and terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) assessments at 2 weeks following the induction of ischemia. Both groups had the same extent of ischemic osteonecrosis and absence of repair at 2 weeks. At 4 weeks, the necrotic epiphyses showed a significant increase in the extent of revascularization in the IL-6 KO mice compared to the wild-type mice (p = 0.001). In addition, a significantly greater recovery of the hematopoietic bone marrow was observed in the osteonecrotic side of the IL-6 KO mice compared to the wild-type mice (p < 0.01). Vascular endothelial growth factor (VEGF) immunohistochemistry showed regionally increased staining in the areas of repair in the osteonecrosis side of IL-6 KO mice compared to the wild-type mice at 4 weeks following ischemic osteonecrosis. Micro-CT assessment of the wild-type mice at 4 weeks showed a significant decrease in the percent bone volume (p < 0.01) in the osteonecrotic side compared to the control side. In contrast, IL-6 KO mice showed significantly increased bone volume in the osteonecrotic side compared to the osteonecrotic side of WT mice (p < 0.001). No significant difference in the bone volume percentage was found between the control side of the wild-type and the IL-6 KO mice. Histomorphometric analysis at 4 weeks revealed increased osteoblast number/bone surface (p < 0.001), bone formation rate (BFR) (p = 0.0001), and mineral apposition rate (MAR) (p < 0.0001) in the osteonecrotic side of the IL-6 KO mice compared to the wild-type mice. The number of osteoclast/bone surface was also increased in the IL-6 KO mice compared to the wild-type mice (p < 0.0001). No significant difference was observed between the control side of the wild-type and IL-6 KO mice with regards to the number of osteoblast or osteoclast/bone surface, BFR, and MAR. We next obtained primary osteoblasts from IL-6 KO mice and showed they expressed a significantly higher level of RANKL/OPG than wild-type mice (p = 0.001) in hypoxia culture condition. Taken together, the findings indicate that IL-6 deletion stimulates revascularization and new bone formation following ischemic osteonecrosis. This study provides new evidence that therapeutic strategies to block IL-6 may be beneficial for bone healing following ischemic osteonecrosis.

Quantitative MRI Helps to Detect Hip Ischemia: Preclinical Model of Legg-Calvé-Perthes Disease.

Purpose To determine whether quantitative MRI relaxation time mapping techniques can help to detect ischemic injury to the developing femoral head. Materials and Methods For this prospective animal study conducted from November 2015 to February 2018, 10 male 6-week-old piglets underwent an operation to induce complete right femoral head ischemia. Animals were humanely killed at 48 hours (n = 2) or 4 weeks (n = 8) after the operation, and the operated and contralateral-control femoral heads were harvested and frozen. Thawed specimens were imaged at 9.4-T MRI by using T1, T2, T1 in the rotating frame (T1ρ), adiabatic T1ρ, relaxation along a fictitious field (RAFF), and T2* mapping and evaluated with histologic analysis. Paired relaxation time differences between the operated and control femoral heads were measured in the secondary ossification center (SOC), epiphyseal cartilage, articular cartilage, and metaphysis and were analyzed by using a paired t test. Results In the SOC, T1ρ and RAFF had the greatest percent increases in the operated versus control femoral heads at both 48 hours (112% and 72%, respectively) and 4 weeks (74% and 70%, respectively). In the epiphyseal and articular cartilage, T2, T1ρ, and RAFF were similarly increased at both points (range, 24%-49%). At 4 weeks, T2, T1ρ, adiabatic T1ρ, and RAFF were increased in the SOC (P = .004, .018, < .001, and .001, respectively), epiphyseal cartilage (P = .009, .008, .011, and .007, respectively), and articular cartilage (P = .005, .016, .033, and .018, respectively). Histologic assessment identified necrosis in SOC and deep layer of the epiphyseal cartilage at both points. Conclusion T2, T1 in the rotating frame, adiabatic T1 in the rotating frame, and relaxation along a fictitious field maps are sensitive in helping to detect ischemic injury to the developing femoral head. © RSNA, 2018 Online supplemental material is available for this article.

Ischemic femoral head osteonecrosis in a piglet model causes three dimensional decrease in acetabular coverage.

Legg-Calve-Perthes disease (LCPD) is a childhood form of ischemic osteonecrosis marked by development of severe femoral head deformity and premature osteoarthritis. The pathogenesis of femoral head deformity has been studied extensively using a piglet model of ischemic osteonecrosis, however, accompanying acetabular changes have not been investigated. The purpose of this study was to determine if acetabular changes accompany femoral head deformity in a well-established piglet model of LCPD and to define the acetabular changes using three dimensional computed tomography (3-D CT) and modeling. Twenty-four piglets were surgically induced with ischemic osteonecrosis on the right side. The contralateral hip was used as control. At 8 weeks postoperative, pelvi were retrieved and imaged with CT. Custom software was used to measure acetabular morphologic parameters on 3-D CT images. Moderate to severe femoral head deformities were present in all animals. Acetabula with accompanying femoral head deformity had a significant decrease in acetabular version and tilt (p < 0.001) and in coverage angle in the superior, posterior, and inferior quadrants (p < 0.05). These findings indicate that the development of femoral head deformity following ischemic osteonecrosis produces specific and predictable changes to the shape of the acetabulum. Acetabular changes described in patients with LCPD were observed in the piglet model. This model may serve as a valuable tool to elucidate the relationship between femoral head and acetabular deformities. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1173-1177, 2018.

Targeted Disruption of NF1 in Osteocytes Increases FGF23 and Osteoid With Osteomalacia-like Bone Phenotype.

Neurofibromatosis type 1 (NF1, OMIM 162200), caused by NF1 gene mutations, exhibits multi-system abnormalities, including skeletal deformities in humans. Osteocytes play critical roles in controlling bone modeling and remodeling. However, the role of neurofibromin, the protein product of the NF1 gene, in osteocytes is largely unknown. This study investigated the role of neurofibromin in osteocytes by disrupting Nf1 under the Dmp1-promoter. The conditional knockout (Nf1 cKO) mice displayed serum profile of a metabolic bone disorder with an osteomalacia-like bone phenotype. Serum FGF23 levels were 4 times increased in cKO mice compared with age-matched controls. In addition, calcium-phosphorus metabolism was significantly altered (calcium reduced; phosphorus reduced; parathyroid hormone [PTH] increased; 1,25(OH)2 D decreased). Bone histomorphometry showed dramatically increased osteoid parameters, including osteoid volume, surface, and thickness. Dynamic bone histomorphometry revealed reduced bone formation rate and mineral apposition rate in the cKO mice. TRAP staining showed a reduced osteoclast number. Micro-CT demonstrated thinner and porous cortical bones in the cKO mice, in which osteocyte dendrites were disorganized as assessed by electron microscopy. Interestingly, the cKO mice exhibited spontaneous fractures in long bones, as found in NF1 patients. Mechanical testing of femora revealed significantly reduced maximum force and stiffness. Immunohistochemistry showed significantly increased FGF23 protein in the cKO bones. Moreover, primary osteocytes from cKO femora showed about eightfold increase in FGF23 mRNA levels compared with control cells. The upregulation of FGF23 was specifically and significantly inhibited by PI3K inhibitor Ly294002, indicating upregulation of FGF23 through PI3K in Nf1-deficient osteocytes. Taken together, these results indicate that Nf1 deficiency in osteocytes dramatically increases FGF23 production and causes a mineralization defect (ie, hyperosteoidosis) via the alteration of calcium-phosphorus metabolism. This study demonstrates critical roles of neurofibromin in osteocytes for osteoid mineralization. © 2017 American Society for Bone and Mineral Research.

Material properties of bone in the femoral head treated with ibandronate and BMP-2 following ischemic osteonecrosis.

Bone morphogenetic protein (BMP)-2 and ibandronate (IB) decrease the femoral head deformity following ischemic osteonecrosis of the femoral head (ONFH). The purpose of this study was to determine the effects of BMP-2 and IB on the mineral content and nanoindentation properties of the bone following ONFH. ONFH was surgically induced in the femoral head of piglets. There were five groups: normal control, untreated, IB, BMP, and BMP + IB (n = 5/group). Backscattered electron imaging, Raman spectroscopy, and nanoindentation testing were performed. Both BMP and BMP + IB groups showed calcium content in the trabecular bone similar to the normal group, while the IB and no-treatment groups showed a significant increase in the calcium content compared to the normal group. The carbonate content relative to phosphate was significantly increased in the IB and BMP + IB groups (p < 0.01) compared to the normal group. No significant difference was found between the BMP and the normal group. The nanoindentation modulus of the bone in the IB group was significantly increased compared to the normal group (p < 0.05). No significant differences were observed between the BMP and BMP + IB groups compared to the normal group. The nanoindentation hardness measurements in the IB group were also significantly increased compared to the BMP and BMP + IB groups (p < 0.05). In summary, trabecular bone treated with BMP or BMP + IB had material properties comparable to normal bone whereas the bone in the IB group retained the increased mineral content and the nanoindentation hardness found in the necrotic bone. Hence, BMP or BMP + IB better restores the normal mineral content and nanomechanical properties after ONFH than IB treatment alone. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1453-1460, 2017.

Necrotic Bone Stimulates Proinflammatory Responses in Macrophages through the Activation of Toll-Like Receptor 4.

In Legg-Calvé-Perthes disease, loss of blood supply results in ischemic osteonecrosis of the femoral head (ONFH). Generally, macrophages play important roles in inflammatory responses to tissue necrosis, but their role in ONFH is not known. The purpose of this study was to determine the macrophage-inflammatory responses after ONFH and the receptor mechanisms involved in sensing the necrotic bone, using a piglet model of Legg-Calvé-Perthes disease. Induction of ONFH resulted in increased numbers of CD14+ macrophages in the fibrovascular repair tissue compared with normal, as determined by immunohistochemistry. Quantitative real-time PCR analysis of macrophages isolated by laser capture microdissection showed significantly increased expression of proinflammatory cytokines IL-1ß, tumor necrosis factor-α, and IL-6 in ONFH compared with normal. Because Toll-like receptors (TLRs) mediate macrophage-inflammatory responses in other inflammatory conditions, we determined their gene expression in macrophages and found significantly increased levels of TLR4 but not TLR2 and TLR9 in ONFH. Mechanistically, in vitro, bone marrow-derived macrophages treated with necrotic bone showed increased extracellular signal-regulated kinases 1/2 and Iκ kinase-α phosphorylation, increased proliferation, migration, and inflammatory cytokine expression, which were blocked by TLR4 inhibitor, TAK242, and by TLR4 ablation in macrophages using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease method. In conclusion, necrotic bone stimulates macrophage-inflammatory responses through TLR4 activation.

Targeted disruption of BMP signaling through type IA receptor (BMPR1A) in osteocyte suppresses SOST and RANKL, leading to dramatic increase in bone mass, bone mineral density and mechanical strength.

Recent studies suggest a critical role of osteocytes in controlling skeletal development and bone remodeling although the molecular mechanism is largely unknown. This study investigated BMP signaling in osteocytes by disrupting Bmpr1a under the Dmp1-promoter. The conditional knockout (cKO) mice displayed a striking osteosclerotic phenotype with increased trabecular bone volume, thickness, number, and mineral density as assessed by X-ray and micro-CT. The bone histomorphometry, H&E, and TRAP staining revealed a dramatic increase in trabecular and cortical bone masses but a sharp reduction in osteoclast number. Moreover, there was an increase in BrdU positive osteocytes (2-5-fold) and osteoid volume (~4-fold) but a decrease in the bone formation rate (~85%) in the cKO bones, indicating a defective mineralization. The SEM analysis revealed poorly formed osteocytes: a sharp increase in cell numbers, a great reduction in cell dendrites, and a remarkable change in the cell distribution pattern. Molecular studies demonstrated a significant decrease in the Sost mRNA levels in bone (>95%), and the SOST protein levels in serum (~85%) and bone matrices. There was a significant increase in the ß-catenin (>3-fold) mRNA levels as well as its target genes Tcf1 (>6-fold) and Tcf3 (~2-fold) in the cKO bones. We also showed a significant decrease in the RANKL levels of serum proteins (~65%) and bone mRNA (~57%), and a significant increase in the Opg mRNA levels (>20-fold) together with a significant reduction in the Rankl/Opg ratio (>95%), which are responsible for a sharp reduction in the cKO osteoclasts. The values of mechanical strength were higher in cKO femora (i.e. max force, displacement, and work failure). These results suggest that loss of BMP signaling specifically in osteocytes dramatically increases bone mass presumably through simultaneous inhibition of RANKL and SOST, leading to osteoclast inhibition and Wnt activation together. Finally, a working hypothesis is proposed to explain how BMPR1A controls bone remodeling by inhibiting cell proliferation and stimulating differentiation. It is reported that RANKL and SOST are abundantly expressed by osteocytes. Thus, BMP signaling through BMPR1A plays important roles in osteocytes.

Bone apatite composition of necrotic trabecular bone in the femoral head of immature piglets.

Ischemic osteonecrosis of the femoral head (IOFH) can lead to excessive resorption of the trabecular bone and collapse of the femoral head as a structure. A well-known mineral component to trabecular bone is hydroxyapatite, which can be present in many forms due to ionic substitution, thus altering chemical composition. Unfortunately, very little is known about the chemical changes to bone apatite following IOFH. We hypothesized that the apatite composition changes in necrotic bone possibly contribute to increased osteoclast resorption and structural collapse of the femoral head. The purpose of this study was to assess the macroscopic and local phosphate composition of actively resorbed necrotic trabecular bone to isolate differences between areas of increased osteoclast resorption and normal bone formation. A piglet model of IOFH was used. Scanning electron microscopy (SEM), histology, X-ray absorbance near edge structure (XANES), and Raman spectroscopy were performed on femoral heads to characterize normal and necrotic trabecular bone. Backscattered SEM, micro-computed tomography and histology showed deformity and active resorption of necrotic bone compared to normal. XANES and Raman spectroscopy obtained from actively resorbed necrotic bone and normal bone showed increased carbonate-to-phosphate content in the necrotic bone. The changes in the apatite composition due to carbonate substitution may play a role in the increased resorption of necrotic bone due to its increase in solubility. Indeed, a better understanding of the apatite composition of necrotic bone could shed light on osteoclast activity and potentially improve therapeutic treatments that target excessive resorption of bone.

Development of a mouse model of ischemic osteonecrosis.

BACKGROUND: Availability of a reliable mouse model of ischemic osteonecrosis could accelerate the development of novel therapeutic strategies to stimulate bone healing after ischemic osteonecrosis; however, no mouse model of ischemic osteonecrosis is currently available. QUESTIONS/PURPOSES: To develop a surgical mouse model of ischemic osteonecrosis, we asked, (1) if the blood vessels that contribute to the blood supply of the distal femoral epiphysis are cauterized, can we generate an osteonecrosis mouse model; (2) what are the histologic changes observed in this mouse model, and (3) what are the morphologic changes in the model. METHODS: We performed microangiography to identify blood vessels supplying the distal femoral epiphysis in mice, and four vessels were cauterized using microsurgical techniques to induce ischemic osteonecrosis. Histologic assessment of cell death in the trabecular bone was performed using terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) and counting the empty lacunae in three serial sections. Quantitation of osteoclast and osteoblast numbers was performed using image analysis software. Morphologic assessments of the distal femoral epiphysis for deformity and for trabecular bone parameters were performed using micro-CT. RESULTS: We identified four blood vessels about the knee that had to be cauterized to induce total ischemic osteonecrosis of the distal femoral epiphysis. Qualitative assessment of histologic sections of the epiphysis showed a loss of nuclear staining of marrow cells, disorganized marrow structure, and necrotic blood vessels at 1 week. By 2 weeks, vascular tissue invasion of the necrotic marrow space was observed with a progressive increase in infiltration of the necrotic marrow space with the vascular tissue at 4 and 6 weeks. TUNEL staining showed extensive cell death in the marrow and trabecular bone 24 hours after the induction of ischemia. The mean percent of TUNEL-positive osteocytes in the trabecular bone increased from 2% ± 1% in the control group to a peak of 98% ± 3% in the ischemic group 1 week after induction of ischemia (mean difference, 96%; 95% CI, 81%-111%; p < 0.0001). The mean percent of empty lacunae increased from 1% ± 1% in the control group to a peak of 78% ± 15% in the ischemic group at 4 weeks (mean difference, 77%; 95% CI, 56%-97%; p < 0.0001). Quantitative analysis showed that the mean number of osteoclasts per bone surface was decreased in the ischemic group at 1, 2, and 4 weeks (p < 0.0001, < 0.0001, and p = 0.02, respectively) compared with the control group. The mean number of osteoclasts increased to a level similar to that of the control group at 6 weeks (p = 0.23). The numbers of osteoblasts per bone surface were decreased in the ischemic group at 1, 2 and 4 weeks (p < 0.0001 for each) compared with the numbers in the control group. The mean number of osteoblasts also increased to a level similar to that of the control group at 6 weeks (p = 0.91). Mean bone volume percent assessed by micro-CT was lower in the ischemic group compared with the control group from 2 to 6 weeks. The mean differences in the percent bone volume between the control and ischemic groups at 2, 4, and 6 weeks were 5.5% (95% CI, 0.9%-10.2%; p = 0.01), 5.3% (95% CI, 0.6%-9.9%; p = 0.02), and 6.0% (95% CI, 1.1%-10.9%; p = 0.008), respectively. A deformity of the distal femoral epiphysis was observed at 6 weeks with the mean epiphyseal height to width ratio of 0.74 ± 0.03 in the control group compared with 0.66 ± 0.06 in the ischemic group (mean difference, 0.08; 95% CI, 0.00-0.16; p = 0.03). CONCLUSION: We developed a novel mouse model of ischemic osteonecrosis that produced extensive cell death in the distal femoral epiphysis which developed a deformity with time. CLINICAL RELEVANCE: The new mouse model may be a useful tool to test potential therapeutic strategies to improve bone healing after ischemic osteonecrosis.

Legg-Calvé-Perthes disease produces chronic hip synovitis and elevation of interleukin-6 in the synovial fluid.

Legg-Calvé-Perthes disease (LCPD) is a childhood hip disorder of ischemic osteonecrosis of the femoral head. Hip joint synovitis is a common feature of LCPD, but the nature and pathophysiology of the synovitis remain unknown. The purpose of this study was to determine the chronicity of the synovitis and the inflammatory cytokines present in the synovial fluid at an active stage of LCPD. Serial MRI was performed on 28 patients. T2-weighted and gadolinium-enhanced MR images were used to assess synovial effusion and synovial enhancement (hyperemia) over time. A multiple-cytokine assay was used to determine the levels of 27 inflammatory cytokines and related factors present in the synovial fluid from 13 patients. MRI analysis showed fold increases of 5.0 ± 3.3 and 3.1 ± 2.1 in the synovial fluid volume in the affected hip compared to the unaffected hip at the initial and the last follow-up MRI, respectively. The mean duration between the initial and the last MRI was 17.7 ± 8.3 months. The volume of enhanced synovium on the contrast MRI was increased 16.5 ± 8.5 fold and 6.3 ± 5.6 fold in the affected hip compared to the unaffected hip at the initial MRI and the last follow-up MRI, respectively. In the synovial fluid of the affected hips, IL-6 protein levels were significantly increased (LCPD: 509 ± 519 pg/mL, non-LCPD: 19 ± 22 pg/mL; p = 0.0005) on the multi-cytokine assay. Interestingly, IL-1ß and TNF-α levels were not elevated. In the active stage of LCPD, chronic hip synovitis and significant elevation of IL-6 are produced in the synovial fluid. Further studies are warranted to investigate the role of IL-6 on the pathophysiology of synovitis in LCPD and how it affects bone healing.

Local administration of bone morphogenetic protein-2 and bisphosphonate during non-weight-bearing treatment of ischemic osteonecrosis of the femoral head: an experimental investigation in immature pigs.

BACKGROUND: Non-weight-bearing decreases the femoral head deformity but increases bone resorption without increasing bone formation in an experimental animal model of Legg-Calvé-Perthes disease. We sought to determine if local administration of bone morphogenetic protein (BMP)-2 with or without bisphosphonate can increase the bone formation during the non-weight-bearing treatment in the large animal model of Legg-Calvé-Perthes disease. METHODS: Eighteen piglets were surgically induced with femoral head ischemia. Immediately following the surgery, all animals received an above-the-knee amputation to enforce local non-weight-bearing (NWB). One to two weeks later, six animals received local BMP-2 to the necrotic head (BMP group), six received local BMP-2 and ibandronate (BMP+IB group), and the remaining six received no treatment (NWB group). All animals were killed at eight weeks after the induction of ischemia. Radiographic, microcomputed tomography (micro-CT), and histomorphometric assessments were performed. RESULTS: Radiographic assessment showed that the femoral heads in the NWB, BMP, and BMP+IB groups had a decrease of 20%, 14%, and 10%, respectively, in their mean epiphyseal quotient in comparison with the normal control group. Micro-CT analyses showed significantly higher femoral head bone volume in the BMP+IB group than in the BMP group (p = 0.02) and the NWB group (p < 0.001). BMP+IB and BMP groups had a significantly higher trabecular number (p < 0.01) and lower trabecular separation (p < 0.02) than the NWB group. In addition, the osteoclast number per bone surface was significantly lower in the BMP+IB group compared with the NWB group. Calcein labeling showed significantly higher bone formation in the BMP and BMP+IB groups than in the NWB group (p < 0.05). Heterotopic ossification was found in the capsule of four hips in the BMP+IB group but not in the BMP group. CONCLUSIONS: Administration of BMP-2 with bisphosphonate best decreased bone resorption and increased new bone formation during non-weight-bearing treatment of ischemic osteonecrosis in a pig model, but heterotopic ossification is a concern. CLINICAL RELEVANCE: This preclinical study provides new evidence that BMP-2 with bisphosphonate can effectively prevent the extreme bone loss associated with the non-weight-bearing treatment and increase new bone formation in the femoral head in this animal model of ischemic osteonecrosis.

Induction of SHP2 deficiency in chondrocytes causes severe scoliosis and kyphosis in mice.

STUDY DESIGN: Genetic engineering techniques were used to develop an animal model of juvenile scoliosis during a postnatal skeletal-growth stage. OBJECTIVE: To investigate the effect of targeted SHP2 (Src homology-2) deficiency in chondrocytes on the development of scoliosis during a juvenile growth stage in mice. SUMMARY OF BACKGROUND DATA: Juvenile idiopathic scoliosis can lead to progressive severe spinal deformity. The pathophysiology and molecular mechanisms responsible for the deformity are unknown. Here, we investigated the role of SHP2 deficiency in chondrocytes as a potential cause of juvenile scoliosis. METHODS: Genetically engineered mice with inducible deletion of SHP2 in chondrocytes were generated. The SHP2 function in chondrocytes was inactivated during a juvenile growth stage from the mouse age of 4 weeks. Radiographical, micro-computed tomographic, and histological assessments were used to analyze spinal changes. RESULTS: When SHP2 deficiency was induced during the juvenile stage, a progressive kyphoscoliotic deformity (thoracic lordosis and thoracolumbar kyphoscoliosis) developed within 2 weeks of the initiation of SHP2 deficiency. The 3-dimensional micro-computed tomography analysis confirmed the kyphoscoliotic deformity with a rotational deformity of the spine and osteophyte formation. The histological analysis revealed disorganization of the vertebral growth plate cartilage. Interestingly, when SHP2 was disrupted during the adolescent to adult stages, no spinal deformity developed. CONCLUSION: SHP2 plays an important role in normal spine development during skeletal maturation. Chondrocyte-specific deletion of SHP2 at a juvenile stage produced a kyphoscoliotic deformity. This new mouse model will be useful for future investigations of the role of SHP2 deficiency in chondrocytes as a mechanism leading to the development of juvenile scoliosis. LEVEL OF EVIDENCE: N/A.