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.

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.

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.

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.

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.

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.