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.

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.

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.

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.

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.

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.

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.

Effects of non-weight-bearing on the immature femoral head following ischemic osteonecrosis: an experimental investigation in immature pigs.

BACKGROUND: Local non-weight-bearing as a treatment for Legg-Calvé-Perthes disease remains controversial since a clear scientific basis for this treatment is lacking. The purpose of this study was to determine the effects of non-weight-bearing on decreasing the femoral head deformity following ischemic osteonecrosis and to investigate its biological effects. METHODS: Unilateral femoral head ischemia was induced in sixteen piglets by placing a ligature around the femoral neck and transecting the ligamentum teres. Eight animals received a hind-limb amputation to prevent weight-bearing on the ischemic side (NWB group). The remaining eight piglets were allowed to bear weight as tolerated (WB group). The contralateral femoral heads of the WB group were used as normal controls. All animals were killed at eight weeks after induction of ischemia, when a deformity is expected in this model. Radiographic, microcomputed tomographic (micro-CT), and histomorphometric assessments were performed. RESULTS: Radiographic and micro-CT assessments showed significantly greater flattening of the infarcted epiphysis in the WB group compared with the NWB group. The mean epiphyseal quotient (ratio of femoral head height to diameter) was significantly lower in the WB group (0.29 ± 0.06) compared with the NWB group (0.41 ± 0.06, p < 0.001). Histomorphometric analyses showed that the mean percentage revascularization of the infarcted epiphysis was significantly greater in the NWB group (95% ± 14%) compared with the WB group (34% ± 33%, p < 0.0004), suggesting that revascularization was more rapid in the NWB group. Both histomorphometric and micro-CT analyses of trabecular bone parameters showed significantly decreased bone volume and decreased trabecular number in the infarcted epiphysis of the NWB group compared with the WB group (p < 0.05). CONCLUSIONS: Local non-weight-bearing decreased the deformity following ischemic femoral head osteonecrosis and increased the rates of revascularization and resorption of the infarcted epiphysis.