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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.

Avascular necrosis (AVN) involves ischemic cell death of the bone. AVN leaves an abundance of necrotic lipids and debris in the bone marrow, which instigates inflammatory bone repair. Consequently, the necrotic bone microenvironment stimulates excessive bone resorption, leading to joint deformities and osteoarthritis. Here, we performed a detergent-assisted bone wash using poloxamer 407 (P407) to clean the necrotic bone environment by removing lipids and necrotic debris. The new concept was tested using an established ex vivo AVN model of porcine cadaver humeral heads. The P407 wash was performed using P407 solution and followed with saline via two intraosseous needles. Visual inspection and image analyses of average pixel light intensity showed that the P407 wash produced a better-cleaned bone than the saline wash. Analyses of the collected bone wash solution showed a two-fold increase in triglycerides (101 vs. 53 mmol/head, p = 0.006) and a 10-fold increase in the dry weight of the removed debris (1.34 vs. 0.13 g/head, p = 0.02) with the P407 wash compared to saline. The histological evaluation showed significantly decreased Oil-Red-O (fats) staining in the P407-washed bone compared with the saline-washed bone. The in vitro assays of Alizarin red and qPCR showed the P407 wash neither altered the osteogenic behaviors of porcine bone marrow-derived mesenchymal cells (pBMMCs) nor raised inflammatory responses of porcine bone marrow-derived macrophages (pBMMs). In conclusion, detergent-assisted bone wash using P407 produced a better removal of nonsoluble debris from the bone marrow space than the saline wash without causing changes to osteogenesis or inflammatory reactions.

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.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).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.The solution collected after each wash showed a significant decrease in the turbidity, cell count, and protein concentration (p0.05). Histologic assessment showed significantly decreased cell debris and adipocytes in the washed group compared to the unwashed group (p0.001).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.III, in vitro comparative study.

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.

Legg-Calve-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).

Introduction Legg–Calve–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 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.

Introduction: Children with Down syndrome (DS) are 10-20 times more likely than children without DS to develop acute lymphoblastic leukemia (ALL), and they demonstrate a distinctive spectrum of genetic alterations. Approximately 50% of DS-ALL cases demonstrate CRLF2 rearrangements (CRLF2-R), an approximately 10-fold higher frequency than in non-DS ALL. We sought to identify the functional basis for the increased incidence of ALL, and specifically CRLF2-R ALL, in children with DS. Methods: We created retroviral vectors which induce overexpression of CRLF2 and green fluorescent protein (GFP) for transduction into bone marrow (BM) cells isolated from the Dp16(1)Yey (Dp16) mouse model of DS, which is trisomic for the approximately 115 human chromosome 21 gene orthologs present on mouse chromosome 16. Transduced BM cells from Dp16 and wild-type (WT) control mice were co-cultured with OP9 stromal cells for one week to promote B-lymphoid lineage development, and then characterized by flow cytometric Hardy fraction analysis, or grown in B-lymphoid-promoting methylcellulose medium for colony growth assays. Results: We achieved efficient transduction (80-95%) of Dp16 and WT BM enriched for hematopoietic stem cells (HSCs) with CRLF2-GFP+ and control GFP+ viruses. Following OP9 co-culture, transduced HSCs were characterized by Hardy fraction analysis. CRLF2-GFP+ Dp16 lymphoid cells demonstrated significantly higher percentages of immature Fraction A (pre-pro-B) cells compared with GFP+ Dp16 cells (39.9% vs 15.7%, p=0.004, Fig. 1A). This CRLF2-GFP-induced immature immunophenotype was more pronounced in Dp16 versus WT HSCs, with a significantly higher percentage of Fraction A cells (39.9% in Dp16 vs 24.0% in WT, p=0.0002) and a significantly lower percentage of more mature Fraction B (pro-B) cells (24.3% in Dp16 vs 49.1% in WT, p=0.02, Fig. 1A,B). In methylcellulose colony assays, CRLF2-GFP+ Dp16 cells yielded a 36-fold increase in B cell colonies compared to GFP+ Dp16 cells (Fig 1C). Again, the effect of CRLF2 transduction was magnified in the Dp16 versus WT background. CRLF2-GFP+ WT cells demonstrated only a 2.9-fold increase in B cell colonies (Fig 1C). Conclusions: Here we demonstrate that CRLF2 overexpression results in a more immature B-lineage immunophenotype and increased lymphoid colony growth in vitro, and that these effects are significantly greater in a murine DS versus WT genetic background. Experiments to investigate the pathways involved and to evaluate these effects in vivo are ongoing. This work provides functional evidence of the enhanced leukemogenicity of CRLF2 overexpression in DS-ALL, and creates a tractable model system for additional future genetic studies. Disclosures No relevant conflicts of interest to declare.