ß-Catenin modulation in neurofibromatosis type 1 bone repair: therapeutic implications.

Tibial pseudarthrosis causes substantial morbidity in patients with neurofibromatosis type 1 (NF1). We studied tibial pseudarthrosis tissue from patients with NF1 and found elevated levels of ß-catenin compared to unaffected bone. To elucidate the role of ß-catenin in fracture healing, we used a surgically induced tibial fracture model in conditional knockout (KO) Nfl (Nf1(flox/flox)) mice. When treated with a Cre-expressing adenovirus (Ad-Cre), there was a localized knockdown of Nf1 in the healing fracture and a subsequent development of a fibrous pseudarthrosis. Consistent with human data, elevated ß-catenin levels were found in the murine fracture sites. The increased fibrous tissue at the fracture site was rescued by local treatment with a Wingless-type MMTV integration site (Wnt) antagonist, Dickkopf-1 (Dkk1). The murine pseudarthrosis phenotype was also rescued by conditional ß-catenin gene inactivation. The number of colony-forming unit osteoblasts (CFU-Os), a surrogate marker of undifferentiated mesenchymal cells able to differentiate to osteoblasts, correlated with the capacity to form bone at the fracture site. Our findings indicate that the protein level of ß-catenin must be precisely regulated for normal osteoblast differentiation. An up-regulation of ß-catenin in NF1 causes a shift away from osteoblastic differentiation resulting in a pseudarthrosis in vivo These results support the notion that pharmacological modulation of ß-catenin can be used to treat pseudarthrosis in patients with NF1.-Ghadakzadeh, S., Kannu, P., Whetstone, H., Howard A., Alman, B. A. ß-catenin modulation in neurofibromatosis type 1 bone repair: therapeutic implications.

MEK-SHP2 inhibition prevents tibial pseudarthrosis caused by NF1 loss in Schwann cells and skeletal stem/progenitor cells.

Congenital pseudarthrosis of the tibia (CPT) is a severe pathology marked by spontaneous bone fractures that fail to heal, leading to fibrous nonunion. Half of patients with CPT are affected by the multisystemic genetic disorder neurofibromatosis type 1 (NF1) caused by mutations in the NF1 tumor suppressor gene, a negative regulator of RAS-mitogen-activated protein kinase (MAPK) signaling pathway. Here, we analyzed patients with CPT and Prss56-Nf1 knockout mice to elucidate the pathogenic mechanisms of CPT-related fibrous nonunion and explored a pharmacological approach to treat CPT. We identified NF1-deficient Schwann cells and skeletal stem/progenitor cells (SSPCs) in pathological periosteum as affected cell types driving fibrosis. Whereas NF1-deficient SSPCs adopted a fibrotic fate, NF1-deficient Schwann cells produced critical paracrine factors including transforming growth factor-ß and induced fibrotic differentiation of wild-type SSPCs. To counteract the elevated RAS-MAPK signaling in both NF1-deficient Schwann cells and SSPCs, we used MAPK kinase (MEK) and Src homology 2 containing protein tyrosine phosphatase 2 (SHP2) inhibitors. Combined MEK-SHP2 inhibition in vivo prevented fibrous nonunion in the Prss56-Nf1 knockout mouse model, providing a promising therapeutic strategy for the treatment of fibrous nonunion in CPT.

Vitamin D concentration in patients with normal and impaired bone union.

BACKGROUND: The aim of the study was to compare vitamin D concentration in patients treated due to delayed bone union and non-union (pseudoarthrosis) and patients with normal fracture healing. MATERIAL/METHODS: A retrospective case-control study was conducted. We enrolled 35 patients with inexplicable (standard and correct surgery, closed fracture, no comorbid metabolic diseases) fracture healing impairment, and 35 patients assigned by age and measurement season. Vitamin D (as 25OHD) concentration was measured in all patients. RESULTS: Vitamin D deficiency was reported in 86% of examined patients. No difference was shown between groups in deficiency prevalence. CONCLUSIONS: Previous studies indicated decreased vitamin D concentration in patients with impaired fracture healing. However, these studies did not include control groups. No difference was demonstrated between patients with normal fracture healing and those with impaired bone union in terms of vitamin D deficiency prevalence.

[The frequency of endothelial dysfunction in patients with pseudarthrosis of long bones with hyperhomocysteinemia and associated metabolic disorder].

In article described research of frequency of endothelial dysfunction in 153 patients with pseudarthrosis of long bones and in individuals with consolidated fractures. The reparative regeneration are associated by structural and functional disorders of the central and peripheral vessels as endothelial dysfunction, thickening of the intima-media, prevails at hypoplastic and atrophic types bone nonunion, neurotrofic syndrome and refractures. Endothelial function was significantly dependent on the levels of homocysteine, total cholesterol and interleukin-6 in serum.

Molecular Dissection of Somatic Skeletal Disease in Neurofibromatosis Type 1.

Neurofibromatosis type 1 (NF1) is a tumor predisposition syndrome caused by heterozygous NF1 gene mutations. Patients with NF1 present with pleiotropic somatic secondary manifestations, including development of bone pseudarthrosis after fracture. Somatic NF1 gene mutations were reproducibly identified in patient-derived pseudarthrosis specimens, suggesting a local mosaic cell population including somatic pathologic cells. The somatic cellular pathogenesis of NF1 pseudarthroses remains unclear, though defects in osteogenesis have been posited. Here, we applied time-series single-cell RNA-sequencing (scRNA-seq) to patient-matched control and pseudarthrosis-derived primary bone stromal cells (BSCs). We show that osteogenic specification to an osteoblast progenitor cell population was evident for control bone-derived cells and haploinsufficient pseudarthrosis-derived cells. Similar results were observed for somatic patient fracture-derived NF1-/- cells; however, expression of genetic pathways associated with skeletal mineralization were significantly reduced in NF1-/- cells compared with fracture-derived NF1+/- cells. In mice, we show that Nf1 expressed in bone marrow osteoprogenitors is required for the maintenance of the adult skeleton. Results from our study implicate impaired Clec11a-Itga11-Wnt signaling in the pathogenesis of NF1-associated skeletal disease. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Glycopeptide bone penetration in patients with septic pseudoarthrosis of the tibia.

BACKGROUND AND OBJECTIVE: In the treatment of bone infections, a major determinant of the clinical response is the active drug concentration at the infected site. Because of the high prevalence of meticillin (methicillin)-resistant staphylococci and enterococci, glycopeptides are widely used for the treatment of bone and joint infections, but data on their penetration into human bone are lacking. The aim of our study was to measure vancomycin and teicoplanin concentrations in infected human bone under steady-state conditions and verify their relationship with inflammatory markers, patient demographic characteristics and pharmacodynamic microbiological markers. METHODS AND PATIENTS: Twenty-seven adult orthopaedic patients undergoing surgical debridement for septic pseudoarthrosis of the tibia and receiving either intravenous vancomycin (Vancocina) 1 g twice daily) or teicoplanin (Targosid) 10 mg/kg/day) were studied from January 2004 to January 2008. Plasma and bone specimens were simultaneously collected during surgery for pharmacokinetic and microbiological assays at a variable interval after antimicrobial administration. Bone samples were dissected into cortical and cancellous bone, cleaned of soft tissues, crushed and eluted into phosphate buffer. Necrotic samples and sequestra were not analysed.Plasma and bone antimicrobial concentrations were measured by a validated method of high-performance liquid chromatography with UV detection, and bone/plasma concentration ratios were calculated. Cortical and cancellous bone area under the concentration-time curve (AUC) over 24 hours (AUC(24)) values were measured by the linear-log trapezoidal rule, using WinNonlin) software, and were compared with the minimum inhibitory concentrations (MICs) of the infecting agents. RESULTS: For vancomycin, the mean +/- SD concentrations were 2.66 +/- 1.2 mg/L in cortical bone and 11.53 +/- 7.8 mg/L in cancellous bone (corresponding to 20.67% and 89.39% of intraoperative plasma concentrations), and the mean +/- SD tissue AUC(24) values were 55.15 +/- 25.26 h . mg/L for cortical bone and 299.16 +/- 299.54 h . mg/L for cancellous bone. For teicoplanin, the mean +/- SD concentrations were 2.01 +/- 1.7 and 7.51 +/- 7.0 mg/L in cortical and cancellous bone, respectively (12.35% and 48.6% of intraoperative plasma concentrations), and the mean +/- SD teicoplanin tissue AUC(24) values were 34.08 +/- 23.6 h . mg/L and 155.17 +/- 132.8 h . mg/L for cortical bone and cancellous bone, respectively. The mean vancomycin AUC(24)/MIC ratios were 215.02 for plasma, 47.14 for cortical bone and 268.95 for cancellous bone. The mean teicoplanin AUC(24)/MIC ratios were 336.48, 36.27 and 197.21 for plasma, cortical bone and cancellous bone, respectively. CONCLUSIONS: Bone penetration of both glycopeptides ranged from poor (<15%) to satisfactory (15-30%) in the cortical compartment, while it was far higher into the highly vascularized cancellous tissue. Vancomycin bone penetration was slightly higher than with teicoplanin, but the difference was not statistically significant. Higher bone concentrations were observed with higher inflammatory markers, possibly as a result of increased vascularization and vascular permeability under inflammatory conditions. Bone concentrations over the MIC and AUC/MIC ratios suggested that both glycopeptides achieve a satisfactory pharmacokinetic exposure in the cancellous bone, as far as Gram-positive pathogens are concerned. On the other hand, cortical bone exposure was suboptimal in most patients. Furthermore, as antimicrobial penetration may be affected by impaired blood supply, the role of radical surgical removal of purulent and necrotic tissues appears to be essential in order to shorten treatment duration and to reduce the risk of treatment failure.

In silico clinical trials for pediatric orphan diseases.

To date poor treatment options are available for patients with congenital pseudarthrosis of the tibia (CPT), a pediatric orphan disease. In this study we have performed an in silico clinical trial on 200 virtual subjects, generated from a previously established model of murine bone regeneration, to tackle the challenges associated with the small, pediatric patient population. Each virtual subject was simulated to receive no treatment and bone morphogenetic protein (BMP) treatment. We have shown that the degree of severity of CPT is significantly reduced with BMP treatment, although the effect is highly subject-specific. Using machine learning techniques we were also able to stratify the virtual subject population in adverse responders, non-responders, responders and asymptomatic. In summary, this study shows the potential of in silico medicine technologies as well as their implications for other orphan diseases.

NF1 gene expression in mouse fracture healing and in experimental rat pseudarthrosis.

Neurofibromatosis type 1 (NF1) is an inherited disease with an incidence of about 1:3000 worldwide. Approximately half of all patients with NF1 present osseous manifestations, which can vary from mild to severely debilitating changes such as congenital pseudarthrosis. In the present study, fracture healing of mouse tibia was followed and specimens were collected 5, 9, 14, and 22 days postoperatively. Experimental pseudarthrosis of rat was followed up to 15 weeks postoperatively. In situ hybridization and immunohistochemistry were used to demonstrate expression of NF1 tumor suppressor and phosphorylated p44/42 mitogen-activated protein kinase (MAPK), an indicator of the Ras-MAPK pathway. The results showed that ossified callus was formed in mouse fracture 22 days after the operation. The final outcome of rat pseudarthrosis was detected 9 weeks after the operation, presenting abundant cartilaginous callus at the pseudarthrosis. NF1 gene expression was noted in the maturing and in the hypertrophic cartilages during normal mouse fracture healing, and in rat pseudarthrosis. Phosphorylated p44/42 MAPK was detected in a subpopulation of the hypertrophic chondrocytes in both models. Furthermore, positive labeling for NF1 mRNA and protein was detected in endothelium in both the pseudarthrosis and in the fracture. In conclusion, NF1 gene expression and function are needed for normal fracture healing, possibly restraining excessive Ras-MAPK pathway activation.

Human pseudoarthrosis tissue contains cells with osteogenic potential.

INTRODUCTION: Nonunion is a challenging problem that may occur after certain bone fractures. The treatment of nonunion is closely related to its type. To develop an effective treatment strategy for each type of nonunion, biological analysis of nonunion tissue is essential. Pseudoarthrosis is a distinct pathologic entity of nonunion. To understand the pathology of pseudoarthrosis, we investigated the cellular properties of pseudoarthrosis tissue-derived cells (PCs) in vitro. PATIENTS AND METHODS: PCs were isolated from four patients with pseudoarthrosis and cultured. Cells were evaluated for cell-surface protein expression by using flow cytometry. Osteogenic differentiation capacity was assessed by using Alizarin Red S staining, alkaline phosphatase (ALP) activity assay, and reverse transcription polymerase chain reaction (RT-PCR) after osteogenic induction. Chondrogenic differentiation capacity was assessed via Safranin O staining and RT-PCR after chondrogenic induction. RESULTS: PCs were consistently positive for the mesenchymal stem cell-related markers CD29, CD44, CD105, and CD166, but were negative for the haematopoietic-lineage markers CD31, CD34, CD45, and CD133. Alizarin Red S staining revealed that PCs formed a mineralised matrix that was rich in calcium deposits after osteogenic induction. ALP activity under osteogenic conditions was significantly higher than that under control conditions. Gene expression of ALP, Runx2, osterix, osteocalcin, and bone sialoprotein was observed in PCs cultured under osteogenic conditions. Induced pellets were negatively stained by Safranin O staining. Gene expression of aggrecan, collagen II, collagen X, SOX5, and SOX9 was not observed. CONCLUSION: We have shown for the first time the properties of cells in patients with pseudoarthrosis. Our results indicated that osteogenic cells existed in the pseudoarthrosis tissue. This study might provide insights into understanding the pathology of pseudoarthrosis and improving the treatment for pseudoarthrosis.

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 TGFß. 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 Nf1osx (-/-) 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.

[Comparative investigations of osteotropic radionucleides. IV. The dynamics of uptake in normal and abnormal bone (author's transl)]. / Vergleichende Untersuchungen mit osteotropen Radionukliden; IV. Dynamik der Anreicherung im normalen und pathologisch veränderten Knochen

The dynamics of uptake of osteotropic radionucleides in normal and abnormal bone were studied by means of sequential and functional scans. Various phosphate and phosphonate complexes were compared in vivo and in vitro. Only phosphonates were considered as suitable for bone scanning. In normal bones in beagles, radioactivity after HEDP fell to 65% after two hours, but was 105% with 18F. In relation to healing fractures, the curves differ quantitatively and qualitatively. In this situation, functional curves derived from dynamic scans provide a better parallel with histological findings than does static scintigraphy with an uptake quotient. Sequential and functional scanning are able to document the therapeutic effect of irradiation of bone metastases.

[Immunohistochemical investigations of congenital pseudarthrosis of the tibia].

OBJECTIVE: To investigate the etiology and pathology of congenital pseudarthrosis. METHOD: Sixty-three specimens were taken from 21 CPT patients. The antibodies, which were used for immunohistochemical studies, were produced against (1) NF-200 or S-100 protein; (2) type I or III collagen; (3) PCNA. The control specimens were taken from neurofibromatosis and traumatic pseudarthrosis. SABC staining was performed. RESULT: Low positive expression of NF-200 (3.2%) and S-100 (4.8%) was noted in the CPT lesion, whereas high expression of NF-200 (85.2%) and S-100 (88.9%) in the lesion of neurofibromatosis. The soft tissues in CPT had a higher ratio of type III to I collagens in comparison with traumatic pseudarthrosis. Positive expression of PCNA (95.2%) was significantly higher in the soft tissues than that (2.5%) in traumatic pseudarthrosis. CONCLUSION: CPT is a disease of non-neuro origin. Its occurrence has no direct relation with neurofibromatosis. CPT has its characteristic pathology in the periosteum rather than in the bone. The pathological fracture or non-union of CPT is caused by fibromatosis, which has a strong activity of cellular proliferation and corrosion. CPT has its pathological property similar to a tumor.

[Tissue metallosis of the implant bed as a late complication of metal osteosynthesis]. / Metalloz tkanei lozha implantata kak pozdnee oslozhnenie metallicheskogo osteosinteza.

Biopsies of soft tissues of 28 patients taken from the region of metallic construction implantation which were in the host for osteosynthesis from 5 months to 4 years were examined histologically, histochemically and electron microscopically. Control biopsis were taken in 4 patients with false joints of long bones 5 months--1 year after the trauma. It is proven that the signs of the metal corrosion destruction are most frequently observed when complex multicomponential metallic implants prepared from heterogeneous metals are used. Corrosion results in the "metallic" tissue response long after the implantation. This is characterized by the formation of dense fibrillar connective tissue, its focal infiltration with fragments of metallic constructions and the products of their destruction in the form of various metal-protein complexes, syderosis, acute or chronic inflammation as a results of corrosion, metallosis and trauma. Metallosis of tissues in the implant bed is to be considered as a late complication of the metallic osteosynthesis.

Neurofibromin deficiency-associated transcriptional dysregulation suggests a novel therapy for tibial pseudoarthrosis in NF1.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease caused by mutations in NF1. Among the earliest manifestations is tibial pseudoarthrosis and persistent nonunion after fracture. To further understand the pathogenesis of pseudoarthrosis and the underlying bone remodeling defect, pseudoarthrosis tissue and cells cultured from surgically resected pseudoarthrosis tissue from NF1 individuals were analyzed using whole-exome and whole-transcriptome sequencing as well as genomewide microarray analysis. Genomewide analysis identified multiple genetic mechanisms resulting in somatic biallelic NF1 inactivation; no other genes with recurring somatic mutations were identified. Gene expression profiling identified dysregulated pathways associated with neurofibromin deficiency, including phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways. Unlike aggressive NF1-associated malignancies, tibial pseudoarthrosis tissue does not harbor a high frequency of somatic mutations in oncogenes or other tumor-suppressor genes, such as p53. However, gene expression profiling indicates that pseudoarthrosis tissue has a tumor-promoting transcriptional pattern, despite lacking tumorigenic somatic mutations. Significant overexpression of specific cancer-associated genes in pseudoarthrosis highlights a potential for receptor tyrosine kinase inhibitors to target neurofibromin-deficient pseudoarthrosis and promote proper bone remodeling and fracture healing.

Adjunctive hyperbaric oxygen therapy in the treatment of atrophic tibial nonunion with Ilizarov external fixator: a radiographic and scintigraphic study in rabbits.

OBJECTIVE: The aim of this experimental study was to determine the effects of adjunctive hyperbaric oxygen therapy (HBO) on atrophic tibial nonunion treatment using Ilizarov external fixator. METHODS: Twenty New Zealand white rabbits were randomly divided into two equal groups. A circular external fixator was applied to the right tibia of all the rabbits. A 5-mm bone block was resected and a tibial pseudarthrosis was obtained after a 6-month waiting period. The experimental group rabbits (n=10) underwent daily 2.5 ATA HBO therapy for 2 hours for 20 days and the control group rabbits (n=10) did not receive any corresponding treatment. Osteoblastic activity was evaluated with bone scintigraphy on days 30 and 90. Fracture healing was evaluated by plain radiographs on days 30 and 90. RESULTS: On Day 30, radiological scores were statistically similar in both groups (p=0.167). However, on Day 90, the experimental group displayed significantly higher radiological scores (p<0.001). Osteoblastic activity was also higher in the experimental group on both scintigraphic assessments (p=0.005 and p=0.001). CONCLUSION: The results of this study suggest that HBO can be used as a supplementary therapy in the management of atrophic tibial nonunion.

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 MyoDnull 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.

Biologic characteristics of fibrous hamartoma from congenital pseudarthrosis of the tibia associated with neurofibromatosis type 1.

BACKGROUND: Fibrous hamartoma is a key pathologic component of congenital pseudarthrosis of the tibia, a challenging and disabling bone disorder. We investigated the biologic characteristics of fibrous hamartoma cells in order to better understand the pathogenesis of this rare disease. METHODS: Fibrous hamartoma tissues were surgically excised at the time of osteosynthesis from seven patients with congenital pseudarthrosis of the tibia associated with neurofibromatosis type 1. Distal tibial periosteum was also harvested as control tissue during tibial derotation osteotomy from two other patients with cerebral palsy and one patient with idiopathic internal tibial torsion. Fibroblast-like cells were enzymatically dissociated and cultured from these tissues. Immunophenotypes were investigated for positive (CD44 and CD105) and negative (CD45 and CD14) mesenchymal lineage cell markers, and the mRNA expressions of bone morphogenetic protein(BMP)-2, BMP-4, and their receptors were assayed by reverse transcription-polymerase chain reaction. After rhBMP-2 treatment, the changes in alkaline phosphatase activity, and in the mRNA expressions of type-I collagen (COL1A1), alkaline phosphatase, and osteocalcin genes, were assayed with use of an RNase protection assay. The mRNA expressions of receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin (OPG) were quantitatively assayed with use of real-time RT-PCR. Osteoclastic differentiation of RAW(264.7) cells in coculture with fibrous hamartoma cells was evaluated. RESULTS: All fibrous hamartoma and tibial periosteal cells tested were CD44+/CD105+/CD45-/CD14- and expressed the mRNAs of BMP-2, BMP-4, and their receptors. The baseline mRNA expressions of COL1A1, alkaline phosphatase, and osteocalcin genes in the fibrous hamartoma cells were diverse. These gene expressions were upregulated by BMP treatment in tibial periosteal cells but did not change or were downregulated in fibrous hamartoma cells. Fibrous hamartoma cells expressed higher levels of RANKL and lower levels of OPG than did tibial periosteal cells. Coculture with fibrous hamartoma cells enhanced osteoclastic differentiation of RAW(264.7) cells. CONCLUSIONS: Fibrous hamartoma cells maintain some of the mesenchymal lineage cell phenotypes, but do not undergo osteoblastic differentiation in response to BMP. They are more osteoclastogenic than are tibial periosteal cells.

The osteogenic potential of pseudoarthrosis tissue and bone from human scaphoid non-unions.

Scaphoid fractures have the highest prevalence of non-union in the human body, but little is known about the osteogenic potential of cells at the pseudoarthrosis. It was our goal to determine whether cells isolated from non-unions could be stimulated to differentiate into osteoblasts and produce bone in vitro. Fifteen human scaphoid non-unions were excised during surgery and bone from either side of the non-union and the fibrocartilagenous central regions were harvested. Osteoblastic populations were subcultured from these. The number of bone nodules (colonies of osteoblast cells that produced bone) from all three regions was similar to the number of nodules derived from iliac bone cultures from the same patients. Treatment of cells with rhBMP-2 resulted in a 3- to 10-fold increase in bone nodule formation in vitro from cells derived from the non-unions. These data demonstrate that cells at the pseudoarthrosis have osteogenic capability and can be stimulated by rhBMP-2, possibly increasing the ability to heal.