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Orthopaedic Proceedings
Vol. 106-B, Issue SUPP_18 | Pages 126 - 126
14 Nov 2024
Lu C Lian W Wu R Lin Y Su C Chen C Tai M Chen Y Wang S Wang F
Full Access

Introduction

Cartilage damage is a critical aspect of osteoarthritis progression, but effective imaging strategies remain limited. Consequently, multimodal imaging approaches are receiving increased attention. Gold nanomaterials, renowned for their therapeutic and imaging capabilities, hold promise in drug development. However, their potential for cartilage imaging is rarely discussed. Here, we developed a versatile nanomaterial, AuNC@BSA-Gd-I, for cartilage detection. By leveraging electrostatic interactions with sulfated glycosaminoglycans (sGAG), the AuNC@BSA-Gd-I can effectively penetrate damaged cartilage while accumulating minimally in healthy cartilage. This probe can be visualized or detected using CT, MRI, IVIS, and a gamma counter, providing a comprehensive approach to cartilage imaging. Additionally, we compared the imaging abilities, cartilage visualization capacities, and versatility of currently disclosed multimodal gold nanomaterials with those of AuNC@BSA-Gd-I.

Method

The physicochemical properties of nanomaterials were measured. The potential for cartilage visualization of these nanomaterials was assessed using an in vitro porcine model. The sGAG content in cartilage was determined using the dimethylmethylene blue (DMMB) assay to establish the correlation between sGAG concentration and imaging intensity acquired at each modality.


Bone & Joint Research
Vol. 13, Issue 2 | Pages 52 - 65
1 Feb 2024
Yao C Sun J Luo W Chen H Chen T Chen C Zhang B Zhang Y

Aims

To investigate the effects of senescent osteocytes on bone homeostasis in the progress of age-related osteoporosis and explore the underlying mechanism.

Methods

In a series of in vitro experiments, we used tert-Butyl hydroperoxide (TBHP) to induce senescence of MLO-Y4 cells successfully, and collected conditioned medium (CM) and senescent MLO-Y4 cell-derived exosomes, which were then applied to MC3T3-E1 cells, separately, to evaluate their effects on osteogenic differentiation. Furthermore, we identified differentially expressed microRNAs (miRNAs) between exosomes from senescent and normal MLO-Y4 cells by high-throughput RNA sequencing. Based on the key miRNAs that were discovered, the underlying mechanism by which senescent osteocytes regulate osteogenic differentiation was explored. Lastly, in the in vivo experiments, the effects of senescent MLO-Y4 cell-derived exosomes on age-related bone loss were evaluated in male SAMP6 mice, which excluded the effects of oestrogen, and the underlying mechanism was confirmed.


Bone & Joint Research
Vol. 12, Issue 8 | Pages 455 - 466
1 Aug 2023
Zhou H Chen C Hu H Jiang B Yin Y Zhang K Shen M Wu S Wang Z

Aims

Rotator cuff muscle atrophy and fatty infiltration affect the clinical outcomes of rotator cuff tear patients. However, there is no effective treatment for fatty infiltration at this time. High-intensity interval training (HIIT) helps to activate beige adipose tissue. The goal of this study was to test the role of HIIT in improving muscle quality in a rotator cuff tear model via the β3 adrenergic receptor (β3AR).

Methods

Three-month-old C57BL/6 J mice underwent a unilateral rotator cuff injury procedure. Mice were forced to run on a treadmill with the HIIT programme during the first to sixth weeks or seventh to 12th weeks after tendon tear surgery. To study the role of β3AR, SR59230A, a selective β3AR antagonist, was administered to mice ten minutes before each exercise through intraperitoneal injection. Supraspinatus muscle, interscapular brown fat, and inguinal subcutaneous white fat were harvested at the end of the 12th week after tendon tear and analyzed biomechanically, histologically, and biochemically.


Bone & Joint Research
Vol. 12, Issue 3 | Pages 202 - 211
7 Mar 2023
Bai Z Shou Z Hu K Yu J Meng H Chen C

Aims

This study was performed to explore the effect of melatonin on pyroptosis in nucleus pulposus cells (NPCs) and the underlying mechanism of that effect.

Methods

This experiment included three patients diagnosed with lumbar disc herniation who failed conservative treatment. Nucleus pulposus tissue was isolated from these patients when they underwent surgical intervention, and primary NPCs were isolated and cultured. Western blotting, reverse transcription polymerase chain reaction, fluorescence staining, and other methods were used to detect changes in related signalling pathways and the ability of cells to resist pyroptosis.


Bone & Joint Research
Vol. 11, Issue 5 | Pages 292 - 300
13 May 2022
He C Chen C Jiang X Li H Zhu L Wang P Xiao T

Osteoarthritis (OA) is a degenerative disease resulting from progressive joint destruction caused by many factors. Its pathogenesis is complex and has not been elucidated to date. Advanced glycation end products (AGEs) are a series of irreversible and stable macromolecular complexes formed by reducing sugar with protein, lipid, and nucleic acid through a non-enzymatic glycosylation reaction (Maillard reaction). They are an important indicator of the degree of ageing. Currently, it is considered that AGEs accumulation in vivo is a molecular basis of age-induced OA, and AGEs production and accumulation in vivo is one of the important reasons for the induction and acceleration of the pathological changes of OA. In recent years, it has been found that AGEs are involved in a variety of pathological processes of OA, including extracellular matrix degradation, chondrocyte apoptosis, and autophagy. Clearly, AGEs play an important role in regulating the expression of OA-related genes and maintaining the chondrocyte phenotype and the stability of the intra-articular environment. This article reviews the latest research results of AGEs in a variety of pathological processes of OA, to provide a new direction for the study of OA pathogenesis and a new target for prevention and treatment.

Cite this article: Bone Joint Res 2022;11(5):292–300.


Bone & Joint Open
Vol. 3, Issue 3 | Pages 211 - 217
1 Mar 2022
Hsu C Chen C Wang S Huang J Tong K Huang K

Aims

The Coronal Plane Alignment of the Knee (CPAK) classification is a simple and comprehensive system for predicting pre-arthritic knee alignment. However, when the CPAK classification is applied in the Asian population, which is characterized by more varus and wider distribution in lower limb alignment, modifications in the boundaries of arithmetic hip-knee-ankle angle (aHKA) and joint line obliquity (JLO) should be considered. The purposes of this study were as follows: first, to propose a modified CPAK classification based on the actual joint line obliquity (aJLO) and wider range of aHKA in the Asian population; second, to test this classification in a cohort of Asians with healthy knees; third, to propose individualized alignment targets for different CPAK types in kinematically aligned (KA) total knee arthroplasty (TKA).

Methods

The CPAK classification was modified by changing the neutral boundaries of aHKA to 0° ± 3° and using aJLO as a new variable. Radiological analysis of 214 healthy knees in 214 Asian individuals was used to assess the distribution and mean value of alignment angles of each phenotype among different classifications based on the coronal plane. Individualized alignment targets were set according to the mean lateral distal femoral angle (LDFA) and medial proximal tibial angle (MPTA) of different knee types.


Bone & Joint Research
Vol. 10, Issue 8 | Pages 514 - 525
2 Aug 2021
Chen C Kang L Chang L Cheng T Lin S Wu S Lin Y Chuang S Lee T Chang J Ho M

Aims

Osteoarthritis (OA) is prevalent among the elderly and incurable. Intra-articular parathyroid hormone (PTH) ameliorated OA in papain-induced and anterior cruciate ligament transection-induced OA models; therefore, we hypothesized that PTH improved OA in a preclinical age-related OA model.

Methods

Guinea pigs aged between six and seven months of age were randomized into control or treatment groups. Three- or four-month-old guinea pigs served as the young control group. The knees were administered 40 μl intra-articular injections of 10 nM PTH or vehicle once a week for three months. Their endurance as determined from time on the treadmill was evaluated before kill. Their tibial plateaus were analyzed using microcalculated tomography (μCT) and histological studies.


Bone & Joint Research
Vol. 10, Issue 2 | Pages 122 - 133
1 Feb 2021
He CP Jiang XC Chen C Zhang HB Cao WD Wu Q Ma C

Osteoarthritis (OA), one of the most common motor system disorders, is a degenerative disease involving progressive joint destruction caused by a variety of factors. At present, OA has become the fourth most common cause of disability in the world. However, the pathogenesis of OA is complex and has not yet been clarified. Long non-coding RNA (lncRNA) refers to a group of RNAs more than 200 nucleotides in length with limited protein-coding potential, which have a wide range of biological functions including regulating transcriptional patterns and protein activity, as well as binding to form endogenous small interference RNAs (siRNAs) and natural microRNA (miRNA) molecular sponges. In recent years, a large number of lncRNAs have been found to be differentially expressed in a variety of pathological processes of OA, including extracellular matrix (ECM) degradation, synovial inflammation, chondrocyte apoptosis, and angiogenesis. Obviously, lncRNAs play important roles in regulating gene expression, maintaining the phenotype of cartilage and synovial cells, and the stability of the intra-articular environment. This article reviews the results of the latest research into the role of lncRNAs in a variety of pathological processes of OA, in order to provide a new direction for the study of OA pathogenesis and a new target for prevention and treatment.

Cite this article: Bone Joint Res 2021;10(2):122–133.


The Bone & Joint Journal
Vol. 102-B, Issue 8 | Pages 967 - 980
1 Aug 2020
Chou TA Ma H Wang J Tsai S Chen C Wu P Chen W

Aims

The aims of this study were to validate the outcome of total elbow arthroplasty (TEA) in patients with rheumatoid arthritis (RA), and to identify factors that affect the outcome.

Methods

We searched PubMed, MEDLINE, Cochrane Reviews, and Embase from between January 2003 and March 2019. The primary aim was to determine the implant failure rate, the mode of failure, and risk factors predisposing to failure. A secondary aim was to identify the overall complication rate, associated risk factors, and clinical performance. A meta-regression analysis was completed to identify the association between each parameter with the outcome.


Full Access

An established rabbit model was used to preliminarily investigate the effect of acellular triphase, namely bone-cartilage-tendon, scaffold (ATS) sandwiched with autologous bone mesenchymal stem cells (BMSCs) sheets on tendon-bone interface healing. Bone, fibrocartilage and tendon tissue were harvested from the rabbits and sectioned into a book-type scaffold. The scaffolds were decellularized and their characterization was presented. BMSCs were isolated and co-cultured with the scaffolds to verify their cytocompatibility. BMSCs sheets were fabricated and inserted into the book page of the scaffold to construct an autologous BMSCs-sheets/book-type ATS complex. The complex was implated in the right knee of rabbits which operated standard partial patellectomy for TBI regeneration using Imaging, histological and biomechanical examinations.

The bone, fibrocartilage and tendon tissue were sectioned into a book-type scaffold before decellularization. Then we decellularized the above tissue and mostly preserved their microstructure and composition of the natural extracellular matrix, including collagen and proteoglycan. After the physicochemical and biological properties of the book-type ATS were evaluated, autologous BMSCs sheets were inserted into the book page of the scaffold to construct an autologous BMSCs-sheets/book-type ATS implants for TBI regeneration. In addition, the ATS has the advantages of non-toxicity, suitable for cell adhesion and growth as well as low immunogenicity while co-cultured with the BMSCs. At the same time, different scaffolds has the ability to induce the osteogenic, chondrogenic and tenogenic differentiation of BMSCs by immunofluorescence, reverse transcription-polymerase chain reaction and western blot analysis.

To determine the efficacy of the tissue-engineered implants for TBI regeneration, we transplanted it into a rabbit patella-patellar tendon (PPT) injury model, and the rabbits were sacrificed at postoperative week 8 or 16 for the radiological, histological, and mechanical evaluation. Radiologically, Synchrotron radiation micro-computed tomography (SR-μCT) showed that BMSCs/ATS group significantly increased bone area, BV/TV, trabecular thickness and trabecular number at the healing interface as compared with other groups at postoperative week 8 or 16. Histologically, the BMSCs/ATS group showed more woven bone, and a more robust fibrocartilaginous junction with a characteristic matrix rich in proteoglycans was seen at the PPT healing interface in comparison with other groups after 8 weeks. At week 16, the healing interface in 3 groups displayed better remodeling with respect to postoperative week 8. Healing and remodeling at the PPT junction were almost complete, with a resemblance to a healthy BTI consisting of the characteristic 4 zones in all groups. At last, we used biomechanical test as functional parameters to evaluate the quality of tendon-bone healing. Biomechanical testing indicated that BMSCs/ATS group showed significantly higher failure load and stiffness than other groups at postoperative week 8 and 16.

The complex composed of acellular triphase, namely bone-cartilage-tendon, scaffold (ATS) sandwiched with autologous bone mesenchymal stem cells (BMSCs) sheets can simulate the gradient structure of tendon-bone interface, inducing stem cell directional differentiation, so as to promote patella-patellar tendon interface healing effectively after injury.


The Bone & Joint Journal
Vol. 101-B, Issue 12 | Pages 1489 - 1497
1 Dec 2019
Wang J Ma H Chou TA Tsai S Chen C Wu P Chen W

Aims

The aim of this meta-analysis was to compare the outcome of total elbow arthroplasty (TEA) undertaken for rheumatoid arthritis (RA) with TEA performed for post-traumatic conditions with regard to implant failure, functional outcome, and perioperative complications.

Materials and Methods

We completed a comprehensive literature search on PubMed, Web of Science, Embase, and the Cochrane Library and conducted a systematic review and meta-analysis. Nine cohort studies investigated the outcome of TEA between RA and post-traumatic conditions. The preferred reporting items for systematic reviews and meta-analysis (Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA)) guidelines and Newcastle-Ottawa scale were applied to assess the quality of the included studies. We assessed three major outcome domains: implant failures (including aseptic loosening, septic loosening, bushing wear, axle failure, component disassembly, or component fracture); functional outcomes (including arc of range of movement, Mayo Elbow Performance Score (MEPS), and the Disabilities of the Arm, Shoulder and Hand (DASH) questionnaire), and perioperative complications (including deep infection, intraoperative fracture, postoperative fracture, and ulnar neuropathy).


Orthopaedic Proceedings
Vol. 101-B, Issue SUPP_9 | Pages 14 - 14
1 Sep 2019
Steenstra I McIntosh G Chen C D'Elia T Amick B Hogg-Johnson S
Full Access

Purposes and Background

Musculoskeletal disorders are leading causes of work disability. Our purpose was to develop a predictive model in a cohort from 2012 and validate the model in 2016 data.

Methods and Results

Prospectively collected data was used to identify inception cohorts in 2012 (n=1652) and 2016 (n=199). Data from back pain claimants receiving treatment in physiotherapy clinics and the Ontario workers' compensation database were linked. Patients were followed for 1 year.

Variables from a back pain questionnaire and clinical, demographic and administrative factors were assessed for predictive value. The outcome was cumulative number of calendar days receiving wage-replacement benefits.

Cox regression revealed 8 significant predictors of shorter time on benefits in the 2012 cohort: early intervention (HR=1.51), symptom duration < 31 days (HR=0.88), not in construction industry (HR=1.89), high Low Back Outcome Score (HR=1.03), younger age (HR=0.99), higher benefit rate (HR=1.00), intermittent pain (HR=1.15), no sleep disturbance (HR=1.15). The 2012 model c-statistic was 0.73 with a calibration slope of 0.90 (SE=0.19, p=0.61) in the 2016 data, meaning not significantly different. The c-statistic in the 2016 data was 0.69. Median duration on benefits of those with a high risk score was 129 days in 2012 and 45 days in 2016.


The Bone & Joint Journal
Vol. 100-B, Issue 9 | Pages 1220 - 1226
1 Sep 2018
Chiu H Chen C Su T Chen C Hsieh H Hsieh C Shen D

Aims

We aimed to determine the effect of dementia and Parkinson’s disease on one, three and 12-month mortality following surgery for fracture of the hip in elderly patients from an Asian population.

Patients and Methods

Using a random sample of patients taken from the Taiwan National Health Insurance Research Database, this retrospective cohort study analyzed the data on 6626 elderly patients who sustained a fracture of the hip between 1997 and 2012 who had ICD-9 codes within the general range of hip fracture (820.xx). We used Cox regression to estimate the risk of death associated with dementia, Parkinson’s disease or both, adjusting for demographic, clinical, treatment, and provider factors.


Orthopaedic Proceedings
Vol. 100-B, Issue SUPP_5 | Pages 9 - 9
1 Apr 2018
Shao H Chen A Chen C Faizan A Scholl D
Full Access

Purpose

Tibial and femoral component overhang in total knee arthroplasty (TKA) is a source of pain, thus is it important to understand anatomic differences between races to minimize overhang by matching the tibial and femoral shaft axis to the knee articular surface. Thus, this study compared knee morphology between Caucasian and East Asian individuals to determine the optimal placement of tibial and femoral stems.

Methods

A retrospective study was conducted on a matched cohort of 50 East Asians (21F, 29M) and 50 Caucasians (21F, 29M) by age and gender. CT scans were obtained in healthy volunteers using <2mm slices. The distance from the proximal tibial diaphysis axis to the tibial plateau center was measured, and the distance from the distal femoral diaphysis axis to the center of distal femoral articular surface was measured. Tibial measurements were made using Akagi's AP axis and the widest ML diameter, and femoral measurements were based on Whiteside's line and the surgical epicondylar axis.


Orthopaedic Proceedings
Vol. 99-B, Issue SUPP_3 | Pages 102 - 102
1 Feb 2017
Dong N Wang J Chen C Wang A Zhou Y
Full Access

Introduction

Self tapping bone screw has been widely used in the fixation of Arthroplasty implants and bone graft. But the unwanted screw or driver breakage can be a direct result of excessive driving torque due to the thread cutting resistance. Previous studies showed that bone drill bit cutting rake angle was a critical factor and was inversely related to the bone cutting efficiency.1, 2, 3, 4 (Figure 1) However to date there was no data for how the rake angle could influence the performance of self tapping bone screw. The purpose of this study was to investigate the torque generated by the self tapping cortical screw in simulated bone insertion as a function of the screw tip cutting flute rake angle.

Methods

Two 5 mm thick BM5166 polyurethane block were stacked together and drilled through with 2.5mm diameter holes. Five 30mm long 3.5 mm diameter Ti6AL4V alloy self tapping cortical screws with 0°rake angle cutting flutes (Figure 2) were inserted in the holes and driven by the spanner attached to the test machine (Z5.0TN/TC-A-10) with a displacement control of 3 revolutions/min and 30N constant axial loading. The screws were driven into the stacked polyurethane block for 8mm depth. The maximum driving torque was recorded. Procedure was repeated for five same screws but with 7° rake angle cutting flutes. (Figure 2) The driving torqueses were compared. Student t test was performed with confidence level of 95% was assumed.


Orthopaedic Proceedings
Vol. 98-B, Issue SUPP_7 | Pages 121 - 121
1 May 2016
Dong N Wang J Chen C Wang A Zhou Y
Full Access

Objective

The purpose of this study was to investigate how rim poly locking scallop cutting depth could affect the rigidity of acetabular cup.

Materials and Methods

(11) generic FEA models including (5) 50mm OD Ti6Al4VELI hemispherical acetabular shells with thicknesses of 3.0, 3.5, 4.0, 4.5 and 5.0mm, and (6) 4mm thick hemispherical shells with standard rim poly indexing scallops varied in cutting depths from inner diameter of the cup in 1.0, 1.5, 2.0, 2.5, 3.0 and 3.5mm. All cups were analyzed in ANSYS® Workbench™ FEA software with a loading condition of 2000N applied to the cup rim per V15 ISO/TC 150/SC 4 N. Verification was carried out by the physical test of a same generic Ti6Al4VELI 50mmOD and 5mm thick solid hemispherical shell under 2000N rim directed load. The cup deformation was compared with FEA results. The maximum deformation of FEA scalloped cups were compared with that of solid hemispherical cups with different shell thickness.


The Bone & Joint Journal
Vol. 97-B, Issue 3 | Pages 427 - 431
1 Mar 2015
Wu C Hsieh P Fan Jiang J Shih H Chen C Hu C

Fresh-frozen allograft bone is frequently used in orthopaedic surgery. We investigated the incidence of allograft-related infection and analysed the outcomes of recipients of bacterial culture-positive allografts from our single-institute bone bank during bone transplantation. The fresh-frozen allografts were harvested in a strict sterile environment during total joint arthroplasty surgery and immediately stored in a freezer at -78º to -68º C after packing. Between January 2007 and December 2012, 2024 patients received 2083 allografts with a minimum of 12 months of follow-up. The overall allograft-associated infection rate was 1.2% (24/2024). Swab cultures of 2083 allografts taken before implantation revealed 21 (1.0%) positive findings. The 21 recipients were given various antibiotics at the individual orthopaedic surgeon’s discretion. At the latest follow-up, none of these 21 recipients displayed clinical signs of infection following treatment. Based on these findings, we conclude that an incidental positive culture finding for allografts does not correlate with subsequent surgical site infection. Additional prolonged post-operative antibiotic therapy may not be necessary for recipients of fresh-frozen bone allograft with positive culture findings.

Cite this article: Bone Joint J 2015;97-B:427–31.


Orthopaedic Proceedings
Vol. 96-B, Issue SUPP_11 | Pages 20 - 20
1 Jul 2014
Lu H Hu J Zhou J Zeng Z Cao Y Chen C
Full Access

Summary Statement

We successfully delineated the 3D micro morphology of chondrocytes in patella-patellar tendon using IL-XPCT for the first time. Compared with conventional histology, IL-XPCT can not only provide a higher resolution imgaing but also keep the 3D integrity of the specimen.

Introduction

The morphology of the bone-tendon junction was complex and quite different from other organs, which result the injured bone-tendon junction repair process too slowly. To study the micro morphology of the bone-tendon junction in 3D may have a great significant value to revealing the repair mechanisms of this pathological process and accelerating injured bone-tendon junction repair. However, it was hindered by the convention methods such as histologic section. In our study, a novel imaging tool, synchrotron radiation based in-line x-ray phase contrast imaging (IL-XPCT) was used to research the 3D micro morphology of the bone-tendon junction.


The Bone & Joint Journal
Vol. 95-B, Issue 7 | Pages 906 - 910
1 Jul 2013
Lin S Chen C Fu Y Huang P Lu C Su J Chang J Huang H

Minimally invasive total knee replacement (MIS-TKR) has been reported to have better early recovery than conventional TKR. Quadriceps-sparing (QS) TKR is the least invasive MIS procedure, but it is technically demanding with higher reported rates of complications and outliers. This study was designed to compare the early clinical and radiological outcomes of TKR performed by an experienced surgeon using the QS approach with or without navigational assistance (NA), or using a mini-medial parapatellar (MP) approach. In all, 100 patients completed a minimum two-year follow-up: 30 in the NA-QS group, 35 in the QS group, and 35 in the MP group. There were no significant differences in clinical outcome in terms of ability to perform a straight-leg raise at 24 hours (p = 0.700), knee score (p = 0.952), functional score (p = 0.229) and range of movement (p = 0.732) among the groups. The number of outliers for all three radiological parameters of mechanical axis, frontal femoral component alignment and frontal tibial component alignment was significantly lower in the NA-QS group than in the QS group (p = 0.008), but no outlier was found in the MP group.

In conclusion, even after the surgeon completed a substantial number of cases before the commencement of this study, the supplementary intra-operative use of computer-assisted navigation with QS-TKR still gave inferior radiological results and longer operating time, with a similar outcome at two years when compared with a MP approach.

Cite this article: Bone Joint J 2013;95-B:906–10.


Orthopaedic Proceedings
Vol. 94-B, Issue SUPP_XXXVIII | Pages 4 - 4
1 Sep 2012
Chen C Uludag H Wang Z Jiang H
Full Access

Purpose

The data regarding the effects of noggin on bone morphogenetic protein (BMP)-induced osteogenesis of mesenchymal stem cells (MSCs) are controversial. Most studies performed in rodent cells/models indicated that noggin was a negative regulator of BMP-2-induced osteogenesis; however, one study conducted with human MSCs in culture showed that the addition of noggin induced osteogenesis in vitro. To clear the controversy, we designed this study to evaluate the effects of knocking down noggin gene expression on BMP-2-induced osteogenesis of human bone marrow-derived primary MSCs in vitro.

Method

MSCs were isolated from human tibial bone marrow by density gradient centrifugation. Two noggin small interfering RNAs (siRNAs) were used in this study to knockdown noggin gene expression. There were four study groups: MSCs with no transfection of siRNA (named as NT group), MSCs transfected with non-targeting negative control siRNA (named as control group), MSCs transfected with noggin siRNA1 (named as NOGsi1 group), and MSCs transfected with noggin siRNA2 (named as NOGsi2 group). After transfection, MSCs were induced to undergo osteogenic differentiation by incubating in basal medium containing 0.1 μg/ml BMP-2 for 35 days. The expression levels of osteoblastic marker genes were measured by real-time quantitative PCR on day 14. Also assessed was alkaline phosphatase (ALP) activity by a colorimetric kinetic assay and Fast Blue B staining on day 14. Calcium deposition was determined by the calcium assay on day 35.