Olumide Aruwajoye

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.

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

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.

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‐1β 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.

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 β-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.

Microcrack density and nanomechanical properties in the subchondral region of the immature piglet femoral head following ischemic osteonecrosis

Development of a subchondral fracture is one of the earliest signs of structural failure of the immature femoral head following ischemic osteonecrosis, and this eventually leads to a flattening deformity of the femoral head. The mechanical and mineralization changes in the femoral head preceding subchondral fracture have not been elucidated. We hypothesized that ischemic osteonecrosis leads to early material and mechanical alterations in the bone of the subchondral region. The purpose of this investigation was to assess the bone of the subchondral region for changes in the histology of bone cells, microcrack density, mineral content, and nanoindentation properties at an early stage of ischemic osteonecrosis in a piglet model. This large animal model has been shown to develop a subchondral fracture and femoral head deformity resembling juvenile femoral head osteonecrosis. The unoperated, left femoral head of each piglet (n=8) was used as a normal control, while the right side had a surgical ischemia induced by disrupting the femoral neck vessels with a ligature. Hematoxylin and eosin (H&E) staining and TUNEL assay were performed on femoral heads from 3 piglets. Quantitative backscattered electron imaging, nanoindentation, and microcrack assessments were performed on the subchondral region of both control and ischemic femoral heads from 5 piglets. H&E staining and TUNEL assay showed extensive cell death and an absence of osteoblasts in the ischemic side compared to the normal control. Microcrack density in the ischemic side (3.2±0.79 cracks/mm(2)) was significantly higher compared to the normal side (0.27±0.27 cracks/mm(2)) in the subchondral region (p<0.05). The weighted mean of the weight percent distribution of calcium (CaMean) also was significantly higher in the ischemic subchondral region (p<0.05). Furthermore, the nanoindentation modulus within localized areas of subchondral bone was significantly increased in the ischemic side (16.8±2.7GPa) compared to the normal control (13.3±3.2GPa) (p<0.05). Taken together, these results support the hypothesis that the nanoindentation modulus of the subchondral trabecular bone is increased in the early stage of ischemic osteonecrosis of the immature femoral head and makes it more susceptible to microcrack formation. We postulate that continued loading of the hip joint when there is a lack of bone cells to repair the microcracks due to ischemic osteonecrosis leads to microcrack accumulation and subsequent subchondral fracture.

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.

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.

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)2D 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.

Development of a Large Animal Model of Non-Weight-Bearing

Decreased weight-bearing (WB) is commonly recommended to treat lower limb bone and joint disorders. The availability of a large animal model of decreased or non–weight–bearing (NWB) would be invaluable to study its effects on bone and joint healing. We report here the development of a piglet model of NWB following the induction of ischemic osteonecrosis of the femoral head. The purpose of this study was to assess the feasibility, effectiveness, and complications of various methods to induce decreased or NWB on a hind limb of piglets. The methods used to decrease WB on a hind limb evolved from an Ehmer sling to patellar and/or Achilles tenotomies to below and above knee amputations (AKAs). The Ehmer sling (n=2) was not tolerated by the piglets and tenotomies (n=3) were ineffective in decreasing WB after 4 weeks. A below knee amputation-induced NWB in 4 out of 7 animals but the 3 remaining animals were found to weight bear on the residual limb which developed a pressure sore, and were later converted to an AKA. AKA induced NWB in all animals (n=8). At 8 weeks, a significantly decreased femoral head deformity was observed in the AKA amputation group (n=8) compared with the WB control group (n=8) indicating the protective effect of NWB (P<0.001). A bony overgrowth was observed at the end of the residual bone in all amputated animals. In conclusion, AKA method consistently induced NWB in immature pigs.