We found that adipose stem cells are poorly differentiated into bone and that their ability to differentiate into bone varies from cell line to cell line. The osteogenic differentiation ability of the adipose stem cell lines was distinguished through Alzarin Red Staining, and the cell lines that performed well and those that did not were subjected to RNA-seq analysis. The selected gene GSTT1 (glutathione S-transferase theta-1) gene is a member of a protein superfamily that catalyzes the conjugation of reduced glutathione to a variety of hydrophilic and hydrophobic compounds. The purpose of this study is to treat avascular necrosis and bone defect by improving bone regeneration with adipose stem cells introduced with a new GSTT1 gene related to osteogenic differentiation of adipose stem cells. In addition, the GSTT1 gene has the potential as a genetic marker that can select a specific cell line in the development of an adipose stem cell bone regeneration drug.

Total RNA was extracted from each sample using the TRIzol reagent. Its concentration and purity were determined based on A260 and A260/A280, respectively, using a spectrophotometer. RNA sequencing library of each sample was prepared using a TruSeq RNA Library Prep Kit. RNA-seq experiments were performed for hADSCs. Cells were transfected with either GSTT1 at 100 nM or siControl (scramble control) by electroporation using a 1050 pulse voltage for 30 ms with 2 pulses using a 10 μl pipette tip.

The purpose of this study is to discover genetic markers that can promote osteogenic differentiation of adipose stem cells (hADSCs) through mRNA-seq gene analysis. The selected GSTT1 gene was found to be associated with the enhancement of osteogenic differentiation of adipose stem cells. siRNA against GSTT1 reduced osteogenic differentiation of hADSCs, whereas GSTT1 overexpression enhanced osteogenic differentiation of hADSCs under osteogenic conditions.

In this study, GSTT1 transgenic adipose stem cells could be used in regenerative medicine to improve bone differentiation. In addition, the GSTT1 gene has important significance as a marker for selecting adipose stem cells with potential for bone differentiation in the development of a therapeutic agent for bone regeneration cells.

In this study, we developed biocompatible adhesive which enables implanted chondrogenic-enhanced hASCs being strongly fixed to the lesion site of defected cartilage.

The bioengineered mussel adhesive protein (MAP) was produced and purified using a bacterial expression system as previously reported. The cell encapsulated coacervate was formulated with two polyelectrolyte, the MAP and 723kDa hyaluronic acid (HA). MAP formed liquid microdroplets with HA and subsequently gelated into microparticles, which is highly viscous and strongly adhesive.

The MAP with chondro-induced hASCs were implanted on the osteochondral defect created in the patellar groove/condyle of OA-induced rabbits. Rabbits were allocated to three different groups as follows: Group1 – Fibrin only; Group2 – Fibrin with hASCs (1.5×10⁶ chondro-induced hASCs); Group3; MAP with hASCs.

The implanted cells were labeled with a fluorescent dye for in vivo visualization. After 35 days, fluorescent signals were more potently detected for MAP with hASCs group than Fibrin with hASCs group in osteochondral defect model. Moreover, histological assessment showed that MAP with hASCs group had the best healing and covered with hyaline cartilage-like tissue. The staining image shows that MAP with hASCs group were filled with perfectly differentiated chondrocytes. Although Fibrin with hASCs group had better healing than fibrin only group, it was filled with fibrous cartilage which owes its flexibility and toughness. As MAP with hASCs group has higher possibility of differentiating to complete cartilage, Fibrin only group and Fibrin with hASCs group have failed to treat OA by rehabilitating cartilage. In order to clarify the evidence of remaining human cell proving efficacy of newly developed bioadhesive, human nuclear staining was proceeded with sectioned rabbit cartilage tissue. The results explicitly showed MAP with hASCs group have retained more human cells than Fibrin only and Fibrin with hASCs groups.

We investigated the waterproof bioadhesive supporting transplanted cells to attach to defect lengthily in harsh environment, which prevents cells from leaked to other region of cartilage. Collectively, the newly developed bio-adhesive, MAP, could be successfully applied in OA treatment as a waterproof bioadhesive with the capability of the strong adhesion to target defect sites.

Osteochondral (OC) defects of the knee are associated with pain and significant limitation of activity. Studies have demonstrated the therapeutic efficacy of mesenchymal stem cell (MSC) therapies in treating osteochondral defects. There is increasing evidence that the efficacy of MSC therapies may be a result of the paracrine secretion, particularly exosomes. Here, we examine the effects of MSC exosomes in combination with Hyaluronic Acid (HA) as an injectable therapy on functional osteochondral regeneration in a rabbit osteochondral defect model.

Exosomes were purified from human MSC conditioned medium by size fractionation. A circular osteochondral defect of 4.5 mm diameter and 2.5 mm depth was surgically created in the trochlear grooves of 16 rabbit knees. Thereafter, eight knees received three weekly injections of 200 µg of exosomes in one ml of 3% HA, and the remaining eight knees received three weekly injections of one ml of 3% HA only. The rabbits were sacrificed at six weeks. Analyses were performed by macroscopic and histological assessments, and functional competence was analysed via Young Modulus calculation at five different points (central, superior, inferior, medial and lateral) of the repaired osteochondral defect site.

MSC exosomes displayed a modal size of 100 nm and expressed exosome markers (CD81, TSG101 and ALIX). When compared to HA alone, MSC exosomes in combination with HA showed significantly better repair histologically and biomechanically. The Young Modulus was higher in 4 out of the 5 points. In the central region, the Young Modulus of MSC exosome and HA combination therapy was significantly higher: 5.42 MPa [SD=1.19, 95% CI: 3.93–6.90] when compared to HA alone: 2.87 MPa [SD=2.10, 95% CI: 0.26–5.49], p < 0 .05. The overall mean peripheral region was also significantly higher in the MSC exosome and HA combination therapy group: 5.87 MPa [SD=1.19, 95% CI: 4.40–7.35] when compared to HA alone: 2.70 MPa [SD=1.62, 95% CI: 0.79–4.71], p < 0 .05. The inferior region showed a significantly higher Young Modulus in the combination therapy: 7.34 MPa [SD=2.14, 95% CI: 4.68–10] compared to HA alone: 2.92 MPa [SD=0.98, 95% CI: 0.21–5.63], p < 0.05. The superior region showed a significantly higher Young Modulus in the combination therapy: 7.31 MPa [SD=3.29, 95% CI: 3.22–11.39] compared to HA alone: 3.59 MPa [SD=2.55, 95% CI: 0.42–6.76], p < 0.05. The lateral region showed a significantly higher Young Modulus in the combination therapy: 8.05 MPa [SD=2.06, 95% CI: 5.49–10.61] compared to HA alone: 3.56 MPa [SD=2.01, 95% CI: 1.06–6.06], p < 0.05. The medial region showed a higher Young Modulus in the combination therapy: 6.68 MPa [SD=1.48, 95% CI: 4.85–8.51] compared to HA alone: 3.45 MPa [SD=3.01, 95% CI: −0.29–7.19], but was not statistically significant. No adverse tissue reaction was observed in all the immunocompetent animals treated with MSC exosomes.

Three weekly injections of MSC exosomes in combination with HA therapy results in a more functional osteochondral regeneration as compared to HA alone.

Adult articular cartilage mechanical functionality is dependent on the unique zonal organization of its tissue. Current mesenchymal stem cell (MSC)-based treatment has resulted in sub-optimal cartilage repair, with inferior quality of cartilage generated from MSCs in terms of the biochemical content, zonal architecture and mechanical strength when compared to normal cartilage. The phenotype of cartilage derived from MSCs has been reported to be influenced by the microenvironmental biophysical cues, such as the surface topography and substrate stiffness. In this study, the effect of nano-topographic surfaces to direct MSC chondrogenic differentiation to chondrocytes of different phenotypes was investigated, and the application of these pre-differentiated cells for cartilage repair was explored.

Specific nano-topographic patterns on the polymeric substrate were generated by nano-thermal imprinting on the PCL, PGA and PLA surfaces respectively. Human bone marrow MSCs seeded on these surfaces were subjected to chondrogenic differentiation and the phenotypic outcome of the differentiated cells was analyzed by real time PCR, matrix quantification and immunohistological staining. The influence of substrate stiffness of the nano-topographic patterns on MSC chondrogenesis was further evaluated. The ability of these pre-differentiated MSCs on different nano-topographic surfaces to form zonal cartilage was verified in in vitro 3D hydrogel culture. These pre-differentiated cells were then implanted as bilayered hydrogel constructs composed of superficial zone-like chondro-progenitors overlaying the middle/deep zone-like chondro-progenitors, was compared to undifferentiated MSCs and non-specifically pre-differentiated MSCs in a osteochondral defect rabbit model.

Nano-topographical patterns triggered MSC morphology and cytoskeletal structure changes, and cellular aggregation resulting in specific chondrogenic differentiation outcomes. MSC chondrogenesis on nano-pillar topography facilitated robust hyaline-like cartilage formation, while MSCs on nano-grill topography were induced to form fibro/superficial zone cartilage-like tissue. These phenotypic outcomes were further diversified and controlled by manipulation of the material stiffness. Hyaline cartilage with middle/deep zone cartilage characteristics was derived on softer nano-pillar surfaces, and superficial zone-like cartilage resulted on softer nano-grill surfaces. MSCs on stiffer nano-pillar and stiffer nano-grill resulted in mixed fibro/hyaline/hypertrophic cartilage and non-cartilage tissue, respectively. Further, the nano-topography pre-differentiated cells possessed phenotypic memory, forming phenotypically distinct cartilage in subsequent 3D hydrogel culture. Lastly, implantation of the bilayered hydrogel construct of superficial zone-like chondro-progenitors and middle/deep zone-like chondro-progenitors resulted in regeneration of phenotypically better cartilage tissue with higher mechanical function.

Our results demonstrate the potential of nano-topographic cues, coupled with substrate stiffness, in guiding the differentiation of MSCs to chondrocytes of a specific phenotype. Implantation of these chondrocytes in a bilayered hydrogel construct yielded cartilage with more normal architecture and mechanical function. Our approach provides a potential translatable strategy for improved articular cartilage regeneration using MSCs.

Twenty-three patients with thirty hips of slipped capital femoral epiphysis were treated in our department, KK Women's and Children's Hospital, Singapore between 1997 and 2005. Except one patient lost of follow-up, twenty-four SCFEs with more than 2 years (25 to 73 months, average 38.5 months) follow-up were reviewed. This study is to evaluate the effectiveness and outcome of our protocol: Russell traction followed by gentle manipulative reduction with a single screw fixation & spica cast immobilization (for acute-on-chronic cases with unstable and reducible SCFE). In this series, there were 13 boys & 5 girls, mean age 12 year old ranging from 10 to 14 years. Among them 7 were Chinese, 6 Malays & 5 Indians. There were 12 unilateral cases (8 on the left & 4 right, 67%) & 6 bilateral cases (33%), including 2 patients found contralateral SCFE subsequently 1 year postoperatively. Acute-on-chronic SCFE were 16 & chronic SCFE 8. 16 were Grate I & 8 Grate II. Russell traction was on preoperatively with an average of 6 days. Gentle manipulative reduction under general anesthesia was performed in 20 SCFEs (12 GI & 8 GII) and 17 of them were successful. Fixation with a single screw was used for all cases except one hip with 2 screws. Average follow-up was 38.5 months. Good results achieved. All patient were symptom free with good function. No complications of AVN, chondrolysis, screw loosening and reslipping of the affective hips. Our protocol of management for SCFE has been largely successful in term of manipulative reduction and fixation.

This study is to evaluate the effectiveness and outcome of our protocol: Russell traction followed by gentle manipulative reduction with a single screw fixation & spica cast immobilization.

Twenty-three patients with thirty hips of slipped capital femoral epiphysis were treated in our department, KK Women’s and Children’s Hospital, Singapore between 1997 and 2005. Except one patient lost of follow-up, twenty-four SCFEs with more than 2 years follow-up were reviewed. In this series, there were 13 boys & 5 girls, mean age 12 year old ranging from 10 to 14 years. Among them 7 were Chinese, 6 Malays & 5 Indians. There were 12 unilateral cases (8 on the left & 4 right, 67%) & 6 bilateral cases (33%), including 2 patients found contralateral SCFE subsequently 1 year postoperatively. Acute-on-chronic SCFE were 16 & chronic SCFE 8. 16 were Grate I & 8 Grate II. Russell traction was on preoperatively with an average of 6 days. Gentle manipulative reduction under general anesthesia was performed in 20 SCFEs (12 GI & 8 GII) and 17 of them were successful. Fixation with a single screw was used for all cases except one hip with 2 screws.

Average follow-up was 38.5 months. Good results achieved. All patient were symptom free with good function. No complications of AVN, chondrolysis, screw loosening and reslipping of the affective hips.

Our protocol of management for SCFE has been largely successful in term of manipulative reduction and fixation.

This is a safe, simple and effective management.

Introduction: In order to investigate the reparative reaction process in patients with osteonecrosis of the femoral head (ONFH), we performed tartrate resistant acid phosphatase (TRAP) staining for the femoral head retrieved from the patients at the surgery.

Methods: This study included 21 hips in 19 patients. There were 15 women and 4 men who had a mean age of 42 years (range, 22 to 79 years). Associated risk factors included corticosteroids (n=15), alcohol (n=4), and idiopathic (n=1). Radiologic staging according to the Japanese Orthopaedic Classification included one stage 1, six stage 3A (collapse < 3mm), five stage3B (collapse> 3mm), and nine stage 4 (osteoarthritic change). After femoral head samples were fixed in formalin, 15 samples were decalcified by EDTA, while 6 were not decalcified. All samples were stained by Haematoxylin and Eosin and TRAP.

Results: TRAP positive multinucleated cells were existed at not only necrotic trabecular bone but also new appositional trabecular bone. TRAP positive cells were mainly located at the reactive interface (revascularized) zone while they did not exist in the necrotic zone. They were sometimes located at the normal bone marrow area near the retinacula or teres. While subchondral fractures were detected in two stage 3A hips, five stage 3B hips, and three stage 4 hips, TRAP positive cells around the subchondral fracture were detected in only two stage 3B hips and three stage 4 hips. In one stage 1 hip, TRAP positive mononuclear cells were detected around the enlarged adipocytes at the reactive interface zone. In six stage 3A hips, TRAP positive cells were detected at the reactive interface zone just around the subchondral area. In five stage 3B hips, they were detected along the reactive interface zone in the femoral head. In nine stage 4 hips, they were detected through the subchondral area and along the reactive interface zone.

Discussion: The expression mode of TRAP positive cells changes according to the radiologic stages in ONFH, represents the reparative reaction process.

In recent years numerous growth factors acting on musculoskeletal tissues have been identified. This presentation summarizes our experience with IGF1 in the stimulation of growth of the physis and TGF beta in the formation of bone and cartilage.

IGF1 in a carrier, agarose, was instilled in a paraphsyeal region in rabbit tibias. The physeal height was measured over a period of time and was found to have increased in the group treated with IGF1 when compared to the control group. In addition there was delayed closure of the physeal plate.

These findings may have clinical applications in stimulation of physeal growth in small by length discrepancies

A polycaprolactone scaffold impregnated with TGF beta was implanted under the skin, in the muscle and under the periosteum in rabbits. Over a period of time the scaffolds were harvested and subjected to histological analysis with a variety to stains. Formation of bone and cartilage was found in these scaffolds implanted under the periosteum. Subdermal and intramuscular implantation of the scaffolds did not produce the same results. It is postulated that apart from TGF Beta local and environmental factors may play a part in bone and cartilage formation. This model may be useful in creating complex scaffolds in-vivo for subsequent transplantations.

In Clinical practice damage to the growth plate is usually caused by trauma. In neonates and infants, sepsis involving the growth plate may lead to very severe deformities as well as limb length discrepancy. The management for the child with physeal growth arrest depends on the age of the child, the site and the extent of involvement of the physis. The assessment of the extent of involvement of the physis can be made by plain x-rays, tomograms and magnetic resonance imaging. In younger children epiphysiolysis with or without an osteotomy is usually performed. In cases where is there is severe limb length discrepancy additional treatment with limb lengthening is carried out. Children towards the end of growth benefit from a corrective osteotomy. Hemichondrodiatasis is not recommended in younger children as there is a risk of physeal fracture leading to further growth arrest. However it can be used for selected cases towards the end of growth.

Epiphysiolysis with the use of interposition materials such as fat, silastic or cement has been shown to be successful for bony bars occupying less than 30 % of the entire physis. In cases where the physeal injury is more extensive recent experimental work has shown that the use of tissue engineering techniques involving the transfer of cultured chondrocytes or mesenchymal stem cells may produce better results than conventional methods.

Objective: To determine the efficacy and safety of pamidronate combined with intramedullary rodding in improving bone mineralisation and reducing fracture incidence in children with osteogenesis imperfecta (O.I.).

Methods: A prospective pilot, open study was performed in which intravenous pamidronate was administered at 1.5 mg/kg bi-monthly to 12 children with O.I., over 18 – 28 months. The children were serially monitored for symptoms, anthropometric measurements, fracture incidence, biochemical assessments of calcium metabolism, bone mineral density (BMD), serum alkaline phosphatase (ALP), urinary N-telopeptides, and spine X-rays. Intra-medullary rodding of fractures were performed with when there was definite angulation of bones.

Results: The number of fractures decreased from 4 to 0.85 fractures/year during pamidronate therapy (p< 0.05). After 18 months of treatment, there was significant improvement in Areal BMD z scores of the lumbar spine from −2.38 to −1.76 (p < 0.05) and in the Volumetric BMD, which increased from 0.06 to 0.09 g/cm3 (p < 0.05). At 18 months, urine N-telopeptide levels (bone resorption marker), decreased from 439.7 to 222.3 BCE/Cr (p < 0.05), and serum ALP (bone formation marker) from 225.0 to 143.5 U/L (p < 0.05), reflecting reduced bone turnover. This may represent a net reduction in bone resorption, and provides a biochemical explanation for the increase in bone mineralisation. Height standard deviation scores were not affected, and there were no significant adverse effects.

Conclusion: 18 months cyclical pamidronate is effective in improving bone mineralisation, and reducing fracture incidence in O.I. Pamidronate therapy, which was safe, and when combined with intra-medullary rodding, can potentially improve the quality of life by improving mobility and preventing post-fracture deformities, thus offering new hope for children afflicted with OI.

Introduction: Surgery in patients with neuromuscular scoliosis is aimed at improving truncal balance, facilitating sitting, prevention of progression of the curve and preservation of respiratory function.

Patients and Methods: This was a retrospective study of surgical results in a group of 24 patients with minimum follow up of 2 years an average post-operative follow up of 5 years (2–9 years) with neuromuscular scoliosis due to varying aetiologies. The aetiologies included SMA (7), CP (6), Duchenne (5), Congenital Myopathies (3), Spina Bifida (2) and Paraspinal Neuroblastoma (1).

Results: The average age at surgery was 10.6 years, the average duration was 4hrs 25mins with an average blood loss pf 1.1 liters. An average ICU stay was 1–2 days and stay in hospital is 11 days. The curves ranged from 25–103° (average 75.6°) pre-operative and we were able to obtain a correction of 56%. In all but 2 of our patients we were successful in preventing deterioration of respiratory function and all our patients could at least sit without support post-operatively. Our complications included only one deep infection (necessitating implant removal), 1 rod breakage and 2 patients with UTI. There were no significant respiratory complications in post-operation.

Conclusion: Spinal surgery in patients with neuromuscular scoliosis is safe and prevents deterioration of respiratory and improve truncal balance and hence quality of life.

86 children with 87 lateral condyle fractures were reviewed. Excellent clinical outcomes in 88.9% of Type 1 undisplaced or < 2mm displaced fractures treated by simple cast immobilisation.

In the Type 2 displaced fractures (2–3mm) treated by cast immobilisation, the risk of secondary displacement was 44%

Conclusion: Undisplaced or < 2mm displaced fracture can be treated conservatively in plaster immobilisation.

For 2–3mm displaced fracture, we recommend percutaneous pinning or open reduction and Kirschner wire fixation.

For displaced or rotated fractures, the fragment should be reduced anatomically and fixed with K wire until radiological union.

We studied the early cartilage changes in osteoarthritis, examining the most normal appearing articular cartilage from the hips of 17 patients. Normal appearing cartilage from five patients treated for fractures was used as control material. Two different types of clone were found. The first had increased staining for proteoglycan and was thought to have been engaged in the synthesis of matrix. The other type was associated with a severe deficiency of proteoglycan, matrix streaks and evidence of degradation and phagocytosis of matrix components. Immunohistochemistry demonstrated large amounts of chondroitin 4 and 6 sulphate about the synthetic-type clones, and little or no reactivity about the degenerative clones which lay more superficially and were associated with matrix destruction. Clones appeared to be engaged in either matrix synthesis or its destruction. The disease process of osteoarthritis appeared to begin at the surface of the articular cartilage.

We reviewed 27 patients who had supracondylar closing wedge osteotomy for cubitus varus. There were 10 excellent and 12 good results. However, of these 22 patients, 14 had a significant bony prominence over the lateral condylar region caused by lateral displacement of the elbow when closing the osteotomy. This prominence was less obvious in patients who had their osteotomy at a young age, but worse after operations near or after skeletal maturity. This difference appeared to be due to remodelling.

Ten adults were studied two to seven years after resection of a fibula for use as a free vascularised bone graft. Six had no symptoms in the donor leg, four had some aching, weakness or paraesthesia and three had definite weakness of the long toe flexors and extensors. All knees and ankles were clinically and radiologically stable, but the distal fibular remnant was osteoporotic in nine patients. Gait analysis of the donor leg and the contralateral normal leg showed definite differences, which could be attributed to weakness of the deep muscles caused by loss of their normal origin and to the change in load transmission through the fibula.