Dong Yeon Lee

Mobilization of endothelial progenitor cells in fracture healing and distraction osteogenesis

INTRODUCTION Fracture healing and distraction osteogenesis (DO) are unique postnatal bone formation processes, and neovascularization is critically required for successful bone regeneration. We investigated endothelial progenitor cell (EPC) mobilization during bone regeneration, and the possible contribution of EPCs to increased vascularization and new bone formation, especially in DO. METHODS Mouse tibia fracture and rat tibia DO models were used in this study. The proportion of EPCs among the peripheral and splenic mononuclear cells (MNCs) was determined by examining the endothelial lineage staining characteristics and EPC cell surface markers. Messenger RNA expression of molecules related to EPC mobilization and homing at the fracture site were analyzed by ribonuclease protection assay and reverse-transcription polymerase chain reaction. In the rat tibia DO model, we measured blood flow during DO, and determined the distribution of ex vivo-expanded and intravenously-infused EPCs. RESULTS The proportion of EPCs among the peripheral and splenic MNCs increased after fracture, peaked on post-fracture day 3, and returned to basal levels during the healing period. Messenger RNA expression of EPC mobilizing cytokines such as vascular endothelial growth factor (VEGF), stem cell factor, monocyte chemoattractant protein-1, and stromal cell-derived factor-1, were upregulated at the fracture callus. The plasma VEGF levels peaked prior to the increase in the EPC proportion. Adhesion molecules involved in EPC homing were expressed at the fracture callus. In the DO model, the temporal pattern of the increase in the EPC proportion was similar to that in the fracture healing model, but the EPC proportion increased again during the distraction and consolidation phases. The distraction gap was relatively ischemic during the distraction phase and blood flow increased profusely later in the consolidation phase. The number of EPCs homing to the bone regeneration site in the DO model correlated with the number of transplanted EPCs in a dose-dependent manner. CONCLUSIONS These findings suggest that signals from the bone regeneration site mobilize EPCs from the bone marrow into the peripheral circulation. Increased EPC mobilization and homing may contribute to neovascularization and thus to new bone formation in fracture healing and DO.

Clinical and radiological manifestations of osteogenesis imperfecta type V.

We reviewed clinical manifestation of 12 patients from three Korean families. They showed mild to moderate bone fragility, and suggested an autosomal dominant inheritance pattern. Significant intrafamilial phenotype variability was obvious. Clinical, radiological, and histopathologic characteristics that distinguished this subtype from others include ossification of interosseous membrane of the forearm with radial head dislocation, hyperplastic callus formation, no evidence of type I collagenopathy and an abnormal histopathologic pattern. Severity of the interosseous membrane ossification was correlated with increasing age (p<0.01) and the radial head dislocation was thought to be a developmental problem rather than a congenital problem. Four children who had bisphosphonate treatment showed improved bone mineral density, radiological changes, and biochemical responses. Osteogenesis imperfecta type V was a distinctive subtype of osteogenesis imperfecta, which caused mild to moderate disability clinically.

Distraction osteogenesis induces endothelial progenitor cell mobilization without inflammatory response in man

INTRODUCTION Distraction osteogenesis (DO) is a unique postnatal bone formation process, which is characterized by a profuse increase in vascularization. Recently endothelial progenitor cells (EPCs) have been reported to circulate in substantial numbers under physiologic conditions and to contribute to bone regeneration. The authors investigated EPC mobilization in patients undergoing limb lengthening. MATERIALS AND METHODS Thirteen of 24 consecutive patients who underwent limb-lengthening procedures on weight-bearing long bones (femur and tibia) were included in this study. Peripheral blood samples were taken at four different time points from each patient, that is, before operation (pre-Op), 2 or 3 days after osteotomy (early-PO), before the start of distraction (pre-Dist), and at 7 to 14 days after the start of distraction (during-Dist). Numbers of leukocytes and levels of plasma C-reactive protein (CRP) were determined. After isolating mononuclear cells (MNCs) by centrifugation, we performed FACS analysis on freshly isolated MNCs using antibodies to the cell surface markers; CD34, CD133, vascular endothelial growth factor receptor 2 (VEGFR2), and alkaline phosphatase. MNCs were also cultured in endothelial cell growth medium and numbers of EPC colony-forming units were counted. Plasma levels of EPC-mobilizing cytokines, such as, VEGF, SDF-1, and MCP-1, were determined by ELISA. RESULTS Numbers of leukocytes and CRP plasma levels increased significantly during the early-PO period (p<0.01) but were maintained within normal range in the during-Dist period. FACS analysis of freshly isolated MNCs showed that EPC-enriched cell fractions increased after distraction, but that alkaline phosphatase-positive cell numbers were unchanged. Numbers of EPC colony-forming units significantly increased in the during-Dist period (p<0.01). Plasma levels of VEGF and SDF-1 significantly increased in the during-Dist period (p<0.05). In femoral lengthening patients whose healing index was less than 30 days/cm, the number of CFUs was 46.8 in the during-Dist period, whereas it was 12.7 in patients whose healing index was more than 30 days/cm (p=0.088). CONCLUSIONS This study demonstrates a mobilization of EPC population during distraction osteogenesis in human limb-lengthening patients. Distraction strain provoked increases in the plasma levels of EPC-mobilizing cytokines, such as, VEGF and SDF-1. These findings suggest a possibility that therapeutical approaches which modulate EPC mobilization may speed bone healing by angiogenesis-osteogenesis coupling during distraction osteogenesis.

ACVR1 Gene Mutation in Sporadic Korean Patients with Fibrodysplasia Ossificans Progressiva

Fibrodysplasia ossificans progressiva (FOP; OMIM 135100) is a rare but extremely disabling genetic disorder of the skeletal system, and is characterized by the progressive development of ectopic ossification of skeletal muscles and subsequent joint ankylosis. The c.617G>A; p.R206H point mutation in the activin A type I receptor (ACVR1) gene has been reported to be a causative mutation of FOP. In the present study, mutation analysis of the ACVR1 gene was performed in 12 patients diagnosed or suspected to have FOP. All patients tested had a de novo heterozygous point mutation of c.617G>A; p.R206H in ACVR1. Mutation analysis confirmed a diagnosis of FOP in patients with ambiguous features, and thus, could be used for diagnostic purposes. Early confirmation through mutation analysis would allow medical professionals to advise on the avoidance of provoking events to delay catastrophic flare-ups of ectopic ossifications.

Disturbed Osteoblastic Differentiation of Fibrous Hamartoma Cell from Congenital Pseudarthrosis of the Tibia Associated with Neurofibromatosis Type I

Background Fibrous hamartoma is the key pathology of congenital pseudarthrosis of the tibia (CPT), which was shown to have low osteogenicity and high osteoclastogenicity. This study further investigated the mechanism of impaired osteoblastic differentiation of fibrous hamartoma cells. Methods Fibroblast-like cells were obtained from enzymatically dissociated fibrous hamartomas of 11 patients with CPT associated with neurofibromatosis type I (NF1). Periosteal cells were also obtained from the distal tibial periosteum of 3 patients without CPT or NF1 as control. The mRNA levels of Wnt ligands and their canonical receptors, such as Lrp5 and β-catenin, were assayed using reverse transcriptase PCR (RT-PCR). Changes in mRNA expression of osteoblast marker genes by rhBMP2 treatment were assayed using quantitative real time RT-PCR. Changes in mRNA expression of transcription factors specifically involved in osteoblastic differentiation by rhBMP2 treatment was also assayed using quantitative real-time RT-PCR. Results Wnt1 and Wnt3a mRNA expression was lower in fibrous hamartoma than in tibial periosteal cells, but their canonical receptors did not show significant difference. Response of osteoblastic marker gene expression to rhBMP2 treatment showed patient-to-patient variability. Col1a1 mRNA expression was up-regulated in most fibrous hamartoma tissues, osteocalcin was up-regulated in a small number of patients, and ALP expression was down-regulated in most fibrous hamartoma tissues. Changes in mRNA expression of the transcription factors in response to rhBMP2 also showed factor-to-factor and patient-to-patient variability. Dlx5 was consistently up-regulated by rhBMP2 treatment in all fibrous hamartoma tissues tested. Msx2 expression was down-regulated by rhBMP2 in most cases but by lesser extent than control tissue. Runx2 expression was up-regulated in 8 out of 18 fibrous hamartoma tissues tested. Osterix expression was up-regulated in 2 and down-regulated in 3 fibrous hamartoma tissues. Conclusions Congenital pseudarthrosis of the tibia appears to be caused by fibrous hamartoma originating from aberrant growth of Nf1 haploinsufficient periosteal cells, which failed in terminal osteoblastic differentiation and arrested at a certain stage of this process. This pathomechanism of CPT should be targeted in the development of novel therapeutic biologic intervention.

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.

Foot and Ankle Function at Maturity After Ilizarov Treatment for Atrophic-Type Congenital Pseudarthrosis of the Tibia: A Comprehensive Outcome Comparison with Normal Controls.

BACKGROUND The purpose of this study was to evaluate clinical outcomes and the biomechanical function of the foot and ankle at skeletal maturity of patients treated for atrophic-type congenital pseudarthrosis of the tibia (CPT) compared with healthy young adult controls. METHODS Twenty-four patients (mean age of 19.1 years) who had undergone Ilizarov treatment for unilateral atrophic-type CPT were compared with twenty-four controls (mean age of 19.6 years). All participants were evaluated using validated outcome questionnaires, radiographs, physical examination, instrumented motion analysis including a multisegmental foot model, and pedobarographic measurement. RESULTS Within the CPT group, the mean score of the American Orthopaedic Foot & Ankle Society (AOFAS) ankle-hindfoot scale was 89.9 (range, 76 to 100), and the mean score of the Oxford Ankle Foot Questionnaire (OAFQ) was 42.8 (range, 15 to 60). Motion analysis and pedobarographic measurement showed differences in biomechanical function of the foot and ankle on the side affected by CPT: a slower walking speed due to the short stride length; decreased dorsiflexion in hallux motion; increased hindfoot pronation in the presence of forefoot supination; diminished ankle push-off power; delayed time to heel-rise; and decreased forefoot pressure relative to hindfoot pressure. However, sagittal motion of the hindfoot and forefoot on the affected side was relatively well preserved. Subgroup analysis demonstrated no significant differences in terms of clinical outcome scores and most biomechanical parameters between the tibiofibular synostosis group and the intact-fibula group. CONCLUSIONS Children with atrophic-type CPT can obtain satisfactory foot and ankle function at maturity after successful Ilizarov treatment. Early stabilization of the ankle mortise by fibular stabilization and preservation of ankle mobility during and after treatment is thought to be crucial to maintaining function of the ankle in patients with CPT. LEVEL OF EVIDENCE Therapeutic Level III. See Instructions for Authors for a complete description of levels of evidence.

Sex differences in knee joint loading: Cross-sectional study in geriatric population.

This study investigated sex differences in knee biomechanics and investigated determinants for difference in a geriatric population. Age‐matched healthy volunteers (42 males and 42 females, average age 65 years) without knee OA were included in the study. Subjects underwent physical examination on their knee and standing full‐limb radiography for anthropometric measurements. Linear, kinetic, and kinematic parameters were compared using a three‐dimensional, 12‐camera motion capture system. Gait parameters were evaluated and determinants for sex difference were evaluated with multiple regression analysis. Females had a higher peak knee adduction moment (KAM) during gait (p = 0.004). Females had relatively wider pelvis and narrower step width (both p < 0.001). However, coronal knee alignment was not significantly different between the sexes. Multiple regression analysis revealed that coronal alignment (b = 0.014, p < 0.001), step width (b = −0.010, p = 0.011), and pelvic width/height ratio (b = 1.703, p = 0.046) were significant determinants of peak KAM. Because coronal alignment was not different between the sexes, narrow step width and high pelvic width/height ratio of female were the main contributors to higher peak KAM in females. Sex differences in knee biomechanics were present in the geriatric population. Increased mechanical loading on the female knee, which was associated with narrow step width and wide pelvis, may play an important role in future development and progression of OA.