Is double inactivation of the Nf1 gene responsible for the development of congenital pseudarthrosis of the tibia associated with NF1?

The pathogenic mechanism responsible for congenital pseudarthrosis of the tibia (CPT) is not well understood although the possibility of double inactivation of the neurofibromatosis type 1 (Nf1) gene has been suggested. In the present study, loss of heterozygosity was investigated in fibrous hamartoma tissues harvested from 16 patients with CPT associated with NF1 using four genetic markers that span the Nf1 gene. Based on the assumption that a single cell with double inactivation of Nf1 would undergo clonal growth and cause fibrous hamartoma, we investigated clonality in fibrous hamartoma tissues by analyzing X‐chromosome inactivation patterns in 11 female patients. Loss of Nf1 heterozygosity in fibrous hamartoma tissues was observed at one or two genetic markers in 4 out of the 16 patients tested. In clonality assays, 3 of 11 patients showed a clonal growth pattern, 5 a non‐clonal pattern, and 3 were non‐informative. These findings support that double inactivation of the Nf1 gene and subsequent clonal growth could be a pathogenic feature of the fibrous hamartoma tissue at least in some of the CPT but might not be essential requirements of CPT development. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1535–1540, 2012     

A murine model of neurofibromatosis type 1 tibial pseudarthrosis featuring proliferative fibrous tissue and osteoclast‐like cells

Neurofibromatosis type 1 (NF1) is a common genetic condition caused by mutations in the NF1 gene. Patients often suffer from tissue‐specific lesions associated with local double‐inactivation of NF1. In this study, we generated a novel fracture model to investigate the mechanism underlying congenital pseudarthrosis of the tibia (CPT) associated with NF1. We used a Cre‐expressing adenovirus (AdCre) to inactivate Nf1 in vitro in cultured osteoprogenitors and osteoblasts, and in vivo in the fracture callus of Nf1^flox/flox and Nf1^flox/? mice. The effects of the presence of Nf1^null cells were extensively examined. Cultured Nf1^null‐committed osteoprogenitors from neonatal calvaria failed to differentiate and express mature osteoblastic markers, even with recombinant bone morphogenetic protein‐2 (rhBMP‐2) treatment. Similarly, Nf1^null‐inducible osteoprogenitors obtained from Nf1 mouse muscle were also unresponsive to rhBMP‐2. In both closed and open fracture models in Nf1^flox/flox and Nf1^flox/? mice, local AdCre injection significantly impaired bone healing, with fracture union being <50% that of wild type controls. No significant difference was seen between Nf1^flox/flox and Nf1^flox/? mice. Histological analyses showed invasion of the Nf1^null fractures by fibrous and highly proliferative tissue. Mean amounts of fibrous tissue were increased upward of 10‐fold in Nf1^null fractures and bromodeoxyuridine (BrdU) staining in closed fractures showed increased numbers of proliferating cells. In Nf1^null fractures, tartrate‐resistant acid phosphatase–positive (TRAP+) cells were frequently observed within the fibrous tissue, not lining a bone surface. In summary, we report that local Nf1 deletion in a fracture callus is sufficient to impair bony union and recapitulate histological features of clinical CPT. Cell culture findings support the concept that Nf1 double inactivation impairs early osteoblastic differentiation. This model provides valuable insight into the pathobiology of the disease, and will be helpful for trialing therapeutic compounds. © 2012 American Society for Bone and Mineral Research     

先天性胫骨假关节中碱性成纤维细胞生长因子（b—FGF）的免疫组化研究

目的：明确碱性成纤维细胞生长因子（b-FGF)在先天性胫肌假关节（CPT）骨膜中的表达水平，探讨b-FGF在CPT发病中的作用，方法：采用免疫组化方法对21例CPT病人的25个病变骨膜标本中b-FGF的表达进行检测。以正常骨膜（10例），创伤性假关节（TP）骨膜（20例）和纤维瘤病组织（10例）作为对照，结果：所有正常骨膜，创伤性假关节标本b-FGF染色均阴性，15例先天胫骨假关节骨膜和6例纤维瘤病病变组织中b-FGF染色阳性，两者无显著性差异（P＞0．05），正常骨膜及创伤性假关节（TP）骨膜中b-FGF表达水平与先天胫骨假关节骨膜和纤维病病变组织有显著性差异（P＜0．01），结论：b-FGF在先天性胫骨假关节（CPT）发病中起了重要作用。     

Preliminary experience with the combined use of recombinant bone morphogenetic protein and bisphosphonates in the treatment of congenital pseudarthrosis of the tibia

Purpose  

Congenital pseudarthrosis of the tibia (CPT) is a rare but serious disorder in children. No single approach has clearly emerged as superior in terms of operative procedure, fixation, optimal time for surgery or adjunctive pharmaceutical intervention. CPT is frequently associated with neurofibromatosis type 1 (NF1), a condition featuring deficient bone anabolism and excessive catabolism. We have therefore combined the use of bone morphogenetic proteins (BMP) with bisphosphonates (BP) as an adjunct to surgical intervention.     

Recent insights into bone development, homeostasis, and repair in type 1 neurofibromatosis (NF1)

Neurofibromatosis type 1 (NF1) is one of the most common single gene syndromes and is typified by a range of characteristic but variably penetrant manifestations. The orthopaedic manifestations of congenital pseudarthrosis of the tibia (CPT) and scoliosis, along with other skeletal defects including sphenoid wing dysplasia, rib penciling, and gracile bones have been classically associated with NF1. Added to this, several recent studies have reported a high prevalence of osteoporosis or osteopenia in NF1 cohorts. Clues to the underlying molecular and cellular deficiencies that cause these bony defects can be gleaned from genetically modified mouse models of Nf1 gene deficiency. These studies suggest that a variety of different cell lineages may be adversely affected by Nf1 haploinsufficiency or by double inactivation of the Nf1 gene. Osteoblasts, osteoclasts, chondrocytes, fibroblasts, and vascular endothelial cells all express the Nf1 gene product, neurofibromin, and may be functionally compromised when levels are decreased or absent. This paper reviews the current literature on NF1 bone development, homeostatic regulation, and repair, and highlights some emerging themes that may have relevance for managing orthopaedic disorders that can arise in individuals with NF1.     

Management of congenital pseudarthrosis of the tibia with the Ilizarov method in a paediatric population: influence of aetiological factors

Purpose

Our study compared the rates of union achieved with the Ilizarov method in congenital pseudarthrosis of the tibia (CPT) associated with neurofibromatosis type 1 (NF1) or CPT of idiopathic origin in paediatric patients.

Methods

We studied the outcomes of 28 children that were treated for CPT between 2005 and 2013. Group 1 included children (n?=?14, mean age?=?9.7 years) with CPT associated with NF1 while group 2 were CPT cases that had radiographic confirmation of dysplastic lesions in the tibia but lacked clinical NF1 manifestations (n?=?14, mean age?=?8.6 years). There was no statistical difference between the groups regarding their age or number of previous operations per patient. Individual technical solutions were planned for each patient but coaptation of bone fragments and autologous local tissue grafting to achieve a greater bone thickness and contact area at the pseudarthrosis level were mainly used. Refracture-free rate after the first operation, number of re-operations per patient, and union rates in the groups were compared.

Results

Bone union and weight bearing were obtained in all the cases after the first operation. Refracture-free rate was 42.86 % in group 1 and 35.71% in group 2 (no statistical difference, p?>?0.05). Mean number of re-operations per patient was 1.07 and 0.78 respectively (p?>?0.05). Subsequent treatment for refractures with the Ilizarov techniques gained 92.86% of union in both groups at the follow-ups by completion of the study (range, 2–9 years).

Conclusions

The Ilizarov method yields comparable results in the management of CPT associated with NF1 or tibial dysplasia of idiopathic origin in paediatric cases. Further research should focus on the ways to support the Ilizarov method in order to reduce the number of repetitive surgeries or eliminate them.

     

儿童先天性胫骨假关节病变骨膜组织的蛋白质组学研究

目的探讨基于串联质谱标签(Tandem Mass Tags,TMT)的蛋白质组学技术鉴定和分析先天性胫骨假关节(congenital pseudarthrosis of the tibia,CPT)病变骨膜组织中的差异表达蛋白。方法取CPT伴神经纤维瘤病1型(neurofibromatosis type 1,NF1)患者(NF1-CPT组)、CPT不伴NF1患者(非NF1-CPT组)和开放性胫骨骨折患者(对照组)的胫骨骨膜标本。采用基于TMT的蛋白质组学技术,对各组胫骨病变骨膜组织中差异表达的蛋白质进行鉴定和分析。设定倍数变化≥1.5或≤0.66且P值<0.05作为筛选差异表达蛋白的阈值。以在线工具DAVID和STRING生物信息学资源用于生成差异蛋白的GO注释、KEGG(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路富集和蛋白质-蛋白质相互作用(protein-protein interaction,PPI)。结果NF1-CPT组中有347种差异表达的蛋白质,其中212种上调、135种下调;非NF1-CPT组中有467种差异蛋白,其中281种上调、186种下调。NF1-CPT组与非NF1-CPT组共有差异蛋白231种,除HLA-DRB1(在NF1-CPT组中上调而在非NF1-CPT组中下调)外,其余230种差异蛋白在两组中表达趋势相同(上调117种、下调113种)。有116种蛋白质仅在NF1-CPT组中发生改变(上调94种、下调22种),236种蛋白质仅在非NF1-CPT组中改变(上调164种、下调72种)。与非NF1-CPT相比,NF1-CPT组的47种蛋白质变化1.5倍(P<0.05)。结论CPT病变骨膜组织与正常胫骨骨膜组织蛋白表达存在差异,NF1-CPT与非NF1-CPT的病变骨膜蛋白表达也存在差异,有助于对CPT发病机制的理解。     

Models of tibial fracture healing in normal and Nf1‐deficient mice

Delayed union and nonunion are common complications associated with tibial fractures, particularly in the distal tibia. Existing mouse tibial fracture models are typically closed and middiaphyseal, and thus poorly recapitulate the prevailing conditions following surgery on a human open distal tibial fracture. This report describes our development of two open tibial fracture models in the mouse, where the bone is broken either in the tibial midshaft (mid‐diaphysis) or in the distal tibia. Fractures in the distal tibial model showed delayed repair compared to fractures in the tibial midshaft. These tibial fracture models were applied to both wild‐type and Nf1‐deficient (Nf1^+/?) mice. Bone repair has been reported to be exceptionally problematic in human NF1 patients, and these patients can also spontaneously develop tibial nonunions (known as congenital pseudarthrosis of the tibia), which are recalcitrant to even vigorous intervention. pQCT analysis confirmed no fundamental differences in cortical or cancellous bone in Nf1‐deficient mouse tibiae compared to wild‐type mice. Although no difference in bone healing was seen in the tibial midshaft fracture model, the healing of distal tibial fractures was found to be impaired in Nf1^+/? mice. The histological features associated with nonunited Nf1^+/? fractures were variable, but included delayed cartilage removal, disproportionate fibrous invasion, insufficient new bone anabolism, and excessive catabolism. These findings imply that the pathology of tibial pseudarthrosis in human NF1 is complex and likely to be multifactorial. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1053–1060, 2008     

Novel method for the treatment of congenital pseudarthrosis of the tibia using the gastrocnemius flap: A preliminary study

Purpose:Congenital pseudarthrosis of the tibia is a rare disease that is particularly difficult to treat; the most difficult complications include nonunion of the tibia, refracture, and failed surgery. This study aimed to evaluate the efficiency of transposing gastrocnemius flaps for the treatment of congenital pseudarthrosis of the tibia.Methods:Nine patients (aged 6.2 ± 3.6 years) diagnosed with congenital pseudarthrosis of the tibia in our hospital between March 2013 and March 2018 were enrolled. The tibial pseudarthrosis and thickened periosteum were completely removed, and intramedullary nails were used to fix the tibia. Bone harvest from the iliac, mixed with allogenic bone, was filled in the gap created by excision of the pseudarthrosis site and the surrounding periosteum; the gastrocnemius flap was then used to wrap the pseudoarthrosis site. The plaster cast was fixed postoperatively. The tibial union was evaluated via radiograph, and the plaster cast was removed after 12–24 weeks. Patients began walking approximately 12–14 weeks postoperatively.Results:Anatomical reduction was achieved in all the patients; the mean bone healing time was 10.1 ± 2.1 months. Bone nonunion was observed in one patient, and no neurovascular injury or wound infection occurred. Limb length discrepancy was in the range 3.2 ± 1.8 cm at 1 year and 4.7 ± 2.7 cm at 2 years after surgery. Two patients underwent replacement of the intramedullary nail, and eight patients exhibited good functional and radiographic outcomes.Conclusion:This preliminary study proved that using the gastrocnemius muscle flap to cover the pseudarthrosis site was an effective method to promote the tibial union and treat congenital pseudarthrosis of the tibia.     

Combined MEK Inhibition and BMP2 Treatment Promotes Osteoblast Differentiation and Bone Healing in Nf1Osx−/− Mice

Neurofibromatosis type I (NF1) is an autosomal dominant disease with an incidence of 1/3000, caused by mutations in the NF1 gene, which encodes the RAS/GTPase‐activating protein neurofibromin. Non‐bone union after fracture (pseudarthrosis) in children with NF1 remains a challenging orthopedic condition to treat. Recent progress in understanding the biology of neurofibromin suggested that NF1 pseudarthrosis stems primarily from defects in the bone mesenchymal lineage and hypersensitivity of hematopoietic cells to TGFβ. However, clinically relevant pharmacological approaches to augment bone union in these patients remain limited. In this study, we report the generation of a novel conditional mutant mouse line used to model NF1 pseudoarthrosis, in which Nf1 can be ablated in an inducible fashion in osteoprogenitors of postnatal mice, thus circumventing the dwarfism associated with previous mouse models where Nf1 is ablated in embryonic mesenchymal cell lineages. An ex vivo–based cell culture approach based on the use of Nf1^flox/flox bone marrow stromal cells showed that loss of Nf1 impairs osteoprogenitor cell differentiation in a cell‐autonomous manner, independent of developmental growth plate–derived or paracrine/hormonal influences. In addition, in vitro gene expression and differentiation assays indicated that chronic ERK activation in Nf1‐deficient osteoprogenitors blunts the pro‐osteogenic property of BMP2, based on the observation that only combination treatment with BMP2 and MEK inhibition promoted the differentiation of Nf1‐deficient osteoprogenitors. The in vivo preclinical relevance of these findings was confirmed by the improved bone healing and callus strength observed in Nf1_osx^?/? mice receiving Trametinib (a MEK inhibitor) and BMP2 released locally at the fracture site via a novel nanoparticle and polyglycidol‐based delivery method. Collectively, these results provide novel evidence for a cell‐autonomous role of neurofibromin in osteoprogenitor cells and insights about a novel targeted approach for the treatment of NF1 pseudoarthrosis. © 2014 American Society for Bone and Mineral Research.     

Hyperactive Transforming Growth Factor‐β1 Signaling Potentiates Skeletal Defects in a Neurofibromatosis Type 1 Mouse Model

Dysregulated transforming growth factor beta (TGF‐β) signaling is associated with a spectrum of osseous defects as seen in Loeys‐Dietz syndrome, Marfan syndrome, and Camurati‐Engelmann disease. Intriguingly, neurofibromatosis type 1 (NF1) patients exhibit many of these characteristic skeletal features, including kyphoscoliosis, osteoporosis, tibial dysplasia, and pseudarthrosis; however, the molecular mechanisms mediating these phenotypes remain unclear. Here, we provide genetic and pharmacologic evidence that hyperactive TGF‐β1 signaling pivotally underpins osseous defects in Nf1^flox/?;Col2.3Cre mice, a model which closely recapitulates the skeletal abnormalities found in the human disease. Compared to controls, we show that serum TGF‐β1 levels are fivefold to sixfold increased both in Nf1^flox/?;Col2.3Cre mice and in a cohort of NF1 patients. Nf1‐deficient osteoblasts, the principal source of TGF‐β1 in bone, overexpress TGF‐β1 in a gene dosage–dependent fashion. Moreover, Nf1‐deficient osteoblasts and osteoclasts are hyperresponsive to TGF‐β1 stimulation, potentiating osteoclast bone resorptive activity while inhibiting osteoblast differentiation. These cellular phenotypes are further accompanied by p21‐Ras–dependent hyperactivation of the canonical TGF‐β1–Smad pathway. Reexpression of the human, full‐length neurofibromin guanosine triphosphatase (GTPase)‐activating protein (GAP)‐related domain (NF1 GRD) in primary Nf1‐deficient osteoblast progenitors, attenuated TGF‐β1 expression levels and reduced Smad phosphorylation in response to TGF‐β1 stimulation. As an in vivo proof of principle, we demonstrate that administration of the TGF‐β receptor 1 (TβRI) kinase inhibitor, SD‐208, can rescue bone mass deficits and prevent tibial fracture nonunion in Nf1^flox/?;Col2.3Cre mice. In sum, these data demonstrate a pivotal role for hyperactive TGF‐β1 signaling in the pathogenesis of NF1‐associated osteoporosis and pseudarthrosis, thus implicating the TGF‐β signaling pathway as a potential therapeutic target in the treatment of NF1 osseous defects that are refractory to current therapies. © 2013 American Society for Bone and Mineral Research.     

先天性胫骨假关节（CPT）的超微结构

目的：深入了解先天性胫骨假关节（CPT）的病理变化、细菌类型及其病变来源。方法：取5例先天性胫骨假关节（CPT）标本中不同部位的组织20例,经固定、切片染色后置于透射电镜下进行观察。结果：（1）断端间组织及病变骨膜组织性质完全一样,均为致密纤维结缔组织,细胞成分多,主要为纤维母细胞、肌纤维母细胞及少量未分化细胞。（2）移行部位骨膜组织结构和病变处骨膜组织结构相似。（3）断端处骨质细胞稀少,部分骨细胞萎缩或坏死,部分骨细胞含有空泡,骨基质未见异常。（4）移行部位骨质未见明显异常表现。结论：（1）先天性胫骨假关节（CPT）是非神经起源的、而是起源于骨膜的一种细胞增生活跃的纤维增生性变;（2）肌纤维母细胞与CPT发病有关。     

Improved union and bone strength in a mouse model of NF1 pseudarthrosis treated with recombinant human bone morphogenetic protein‐2 and zoledronic acid

Tibial pseudarthrosis associated with Neurofibromatosis type 1 (NF1) is an orthopedic condition with consistently poor clinical outcomes. Using a murine model that features localized double inactivation of the Nf1 gene in an experimental tibial fracture, we tested the effects of recombinant human bone morphogenetic protein‐2 (rhBMP‐2) and/or the bisphosphonate zoledronic acid (ZA). Tibiae were harvested at 3 weeks for analysis, at which time there was negligible healing in un‐treated control fractures (7% union). In contrast, rhBMP‐2 and rhBMP‐2/ZA groups showed significantly greater union (87% and 93%, p < 0.01 for both). Treatment with rhBMP‐2 led to a 12‐fold increase in callus bone volume and this was further increased in the rhBMP‐2/ZA group. Mechanical testing of the healed rhBMP‐2 and rhBMP‐2/ZA fractures showed that the latter group had significantly higher mechanical strength and was restored to that of the un‐fractured contralateral leg. Co‐treatment with rhBMP‐2/ZA also reduced fibrous tissue infiltration at the fracture site compared to rhBMP alone (p = 0.068). These data support the future clinical investigation of this combination of anabolic and anti‐resorptive agents for the treatment of NF1 pseudarthrosis. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:930–936, 2018.

     

先天性胫骨假关节的免疫组化研究及临床意义

探讨先天性胫骨假关节发病原因,病变属性等生物学行为,为更合理的临床治疗提供理论依据。方法用２１例ＣＰＴ中不同部位的６３个标本,抗体分别为抗：（１）神经丝蛋白、Ｓ－１００蛋白;（２）Ⅰ、Ⅲ型胶原蛋白;（３）增殖细胞核抗原。以神经纤维瘤病和创伤性假关节作对照。ＳＡＢＣ法染色判定结果。结果ＮＦ２００、要００Ｄ     

Masquelet’s procedure and bone morphogenetic protein in congenital pseudarthrosis of the tibia in children: a case series and meta-analysis

Purpose

A type 2 recombinant human bone morphogenetic protein (rhBMP2) and Masquelet’s procedure were used in three children presenting with congenital pseudarthrosis of the tibia (CPT). Recent studies on CPT suggested the presence in situ of pathologic tissues promoting pseudarthrosis. The authors hypothesized that large segmental resection of pseudarthrosis could improve prognosis of the CPT. Masquelet’s procedure and rhBMP2 have been advocated for the treatment of long bone defect.

Method

The authors report three cases of CPT in children treated with Masquelet’s procedure and application of rhBMP2. They analyzed all published cases of CPT similarly treated.

Results

In the present study, Masquelet’s procedure did not improve the results in the treatment of CPT, but segmental bone reconstruction was possible. Bone healing was obtained in three out of the five applications of rhBMP2. In one case, the patient’s parents asked for leg amputation. Analysis of the 33 published cases with the application of BMP in CPT points to a 62 % healing rate in this pathology.

Conclusion

The authors confirmed that segmental bone reconstruction is possible in CPT using Masquelet’s procedure. In the literature, the success rate of the application of rhBMP in CPT appears to be lower than the healing rate usually reported without BMP. Nevertheless, the strict selection of patients, limited number of cases, and their heterogeneity make interpreting the results difficult. However, the theoretical risk which the children are exposed to during the use of BMP makes rigorous selection of the indications necessary. Finally, the interest of rhBMP2 application in Masquelet’s procedure remained to be proven.     

Combined massive allograft and intramedullary vascularized fibula transfer: the Capanna technique for treatment of congenital pseudarthrosis of the tibia

Background and purpose — Congenital pseudarthrosis of the tibia (CPT) is caused by local periosteal disease that can lead to bowing, fracturing, and pseudarthrosis. Current most successful treatment methods are segmental bone transport and vascularized and non-vascularized bone grafting. These methods are commonly hampered by discomfort, reoperations, and long-term complications. We report a combination of a vascularized fibula graft and large bone segment allograft, to improve patient comfort with similar outcomes.Patients and methods — 7 limbs that were operated on in 6 patients between November 2007 and July 2018 with resection of the CPT and reconstruction with a vascularized fibula graft in combination with a bone allograft were retrospectively studied. The mean follow-up time was 5.4 years (0.9–9.6). Postoperative endpoints: time to discharge, time to unrestricted weight bearing, complications within 30 days, consolidation, number of fractures, and secondary deformities.Results — The average time to unrestricted weight bearing with removable orthosis was 3.5 months (1.2–7.8). All proximal anastomoses consolidated within 10 months (2–10). 4 of the 7 grafts fractured at the distal anastomosis between 6 and 14 months postoperatively. After reoperation, consolidation of the distal anastomosis was seen after 2.8 months (2–4). 1 patient required a below-knee amputation.Interpretation — This case series showed favorable results of the treatment of CPT through a combination of a vascularized fibula graft and large bone segment allograft, avoiding the higher reintervention rate and discomfort with ring frame bone transport, and the prolonged non-weight bearing with vascularized fibula transfer without reinforcement with a massive large bone segment allograft.

Congenital pseudarthrosis of the tibia (CPT) is a rare disease affecting the development of the diaphysis of the tibia with a reported incidence ranging between 1 in 140,000 to 250,000 newborns (Ruggieri and Huson 2001, Horn et al. 2013). CPT is characterized by local periosteal disease, often leading to bowing and fracturing of the tibia and/or fibula, followed by the development of a pseudarthrosis (Stevenson et al. 1999). The etiology behind CPT remains largely unelucidated. Many theories regarding the influence of vascular, genetic, and mechanical factors have been proposed over the years, but none provides an entirely satisfactory explanation for the pathological features or its typical location (Hefti et al. 2000, Hermanns-Sachweh et al. 2005). However, there is a clear association with type 1 neurofibromatosis (NF1), as the prevalence of NF1 in CPT patients exceeds 50% (Van Royen et al. 2016).The challenge in cases of pseudarthrosis in CPT is obtaining solid union of the tibia with minimal limb length discrepancy and angular deformity (Grill et al. 2000). The most used treatments today are resection of the CPT part of the bone and vertical bone transport or the use of a pedicled or free vascularized fibula graft (Kesireddy et al. 2018). A multinational study from Japan, which included both the Ilizarov technique combined with diaphyseal transfer and vascularized fibula grafting, found high rates of union among both treatment groups and concluded that both approaches should be considered (Ohnishi et al. 2005).For the Ilizarov technique with diaphyseal transfer through proximal metaphyseal corticotomy and distraction at a distance from the dystrophic area, success rates between 50% and 90% have been reported (Paley et al. 1992, Ghanem et al. 1997, Romanus et al. 2000, Choi et al. 2011). Drawbacks of this technique are multiple interventions and prolonged discomfort of the patient due to many months of wearing a ring frame around the lower leg with pins and K-wires moving through muscle compartments. Also, pin tract infections and surgery to induce healing at the docking side of the bone transport lead to restrictions in social and psychological functioning (Ramaker et al. 2000, Patterson 2006).Studies on vascularized fibula grafts report union rates up to over 90% (Weiland et al. 1990, Erni et al. 2010). Drawbacks of this method include the prolonged period of non-weight bearing due to the lack of primary mechanical strength, resulting in graft fractures requiring reoperations prior to consolidation and hypertrophy of the graft (Weiland et al. 1990, Bos et al. 1993, Romanus et al. 2000, Ohnishi et al. 2005).In 1993, Capanna et al. reported on his “Capanna technique” in the resection of bone tumors in which the use of a vascularized autograft is combined with the use of a solid bone segment allograft in order to achieve instant stability with solid bone during consolidation and subsequent hypertrophy of the vascularized fibula during growth (Capanna et al. 1993).In order to avoid the drawbacks and discomfort of bone transport or vascularized fibula transfer without adding initial additional stability, we introduced this technique for the treatment of pseudarthrosis in CPT. Paley (2019) recently published the outcomes of his cross-union concept, reporting union in all 17 treated patients without refracturing with his latest technique, with follow-up to 11 years. If these outcomes prove to be reproducible, this will probably make the cross-union technique the gold standard for treating CPT. We report a retrospective case series on the Capanna technique in patients with CPT as reference for future strategies in this disease.     

Relationship of donor site to chondrogenic potential of periosteum in vitro

Periosteum has been shown in vitro and in vivo to have a chondrogenic potential that permits it to be used for cartilage regeneration. A useful donor site should have good chondrogenic potential, availability of a large quantity of periosteum, and relative ease of access, and it should be associated with a low rate of morbidity. We hypothesized that the chondrogenic potential of periosteum varies from one bone to another and among different regions of the periosteum from a single bone. A total of 370 periosteal and 37 fascia lata (control) explants were taken from the skull, the ilium, the scapula, the upper, middle, and lower medial proximal tibia, the posterior proximal tibia, and the distal tibia of 2-month-old New Zealand rabbits. The explants were cultured for 6 weeks in agarose/Dulbecco's modified Eagle medium to which 10 ng/ml of transforming growth factor-β1 was added during the first 2 weeks. Skeletal muscle and fascia lata were used as controls. In addition, the thickness, cell density, and total cell count of the cambium layer were measured in 24 explants from the donor sites on the ilium and the upper, middle, and lower proximal tibia. At 6 weeks, histomorphometry and quantitative collagen typing were performed. The periosteal donor sites could be grouped into three categories according to chondrogenic potential: ilium (best), scapula and tibia, and skull (no chondrogenesis). The scapular periosteum was slightly better than that from the tibia. Within the tibia, the upper and middle zones of the proximal region were similar and were slightly better than the lower proximal tibia or the distal tibia. The cellularity of the cambium layer correlated positively with the amount of cartilage as a percentage of the total area. The results of this study indicate that iliac periosteum exhibited the best overall chondrogenic potential in vitro but that periosteum from the traditionally used medial proximal tibia also was excellent. Periosteum from the skull was not chondrogenic. The chondrogenic potential of periosteum varies from bone to bone and within the periosteum from one bone. This variation in chondrogenic potential among donor sites may be due to a difference in the total cell count of the cambium layer.     

NF1 tumor suppressor protein and mRNA in skeletal tissues of developing and adult normal mouse and NF1-deficient embryos.

NF1 is a heritable disease with multiple osseous lesions. The expression of the NF1 gene was studied in embryonic and adult rodent skeleton and in NF1-deficient embryos. The NF1 gene was expressed intensely in the cartilage and the periosteum. Impaired NF1 expression may lead to inappropriate development and dynamics of bones and ultimately to the osseous manifestations of the disease. INTRODUCTION: Neurofibromatosis type 1 is caused by mutations in the NF1 gene encoding the Ras GTPase activating protein (Ras-GAP) neurofibromin. Skeletal ailments such as short stature, kyphoscoliosis, and tibial bowing and pseudarthrosis are common osseous manifestations of NF1. These symptoms are congenital, implying a role for neurofibromin in proper bone growth. However, little is known about its expression in skeletal tissues during their development. MATERIALS AND METHODS: The expression of the NF1 gene was studied in normal and NF1+/- mouse fetuses at embryonic days 12.5-15.5 and in skeletal tissues of adult mice and rats. In situ hybridization, immunohistochemistry, and Western blot analysis were used to identify the NF1 gene expression profile. RESULTS: NF1 mRNA and protein were elevated in resting, maturation, and hypertrophic chondrocytes at the growth plate. Parallel studies on NF1+/- embryos showed expression patterns identical to wildtype. The periosteum, including osteoblasts and osteoclasts, and osteocytes of the cortical bone of adult mice were also intensely labeled for NF1 protein and mRNA. Western transfer analysis detected NF1 protein in the respective rat tissues. Phosphorylation of p42 and p44 MAP kinases, the downstream consequence of Ras activation, was elevated in hypertrophic chondrocytes of NF1+/- embryos. CONCLUSIONS: The results suggest that neurofibromin may act as a Ras-GAP in skeletal cells to attenuate Ras transduced growth signals and thus play a role during ossification and dynamics of bone. Loss of NF1 function may therefore lead to dysplastic bone growth, thereby causing the debilitating osseous symptoms of NF1.