The Effects of Extracellular pH on Proliferation and Differentiation of human Bone Marrow Stem Cells

OBJECTIVES: The purpose of this study is to identify whether the change of pH affects the proliferation and the differentiation of human bone marrow stem cells (hBMSCs) and what mechanism is underlied. METHODS: To achieve objective of this study, hBMSCs were cultivated in the conditioned media adjusted to potential of hydrogen (pH) ranging from 6.4 to 8.0 using addition of hydrochloric acid (HCl) and sodium hydroxide (NaOH). The ratio of proliferation of hBMSCs according to the change of pH was measured for 24 h, 48 h, and 72 h using water-soluble tetrazolium salt (WST)-8 method. To elucidate the mechanism involved, hBMSCs was subjected to blocking extracellular signal-regulated kinases (ERK) and calcium sensing receptor (CaSR) activation. The Osteogenic-related genes and alkaline phosphatase (ALP) activity were tested under the conditioned media. RESULTS: The proliferation of hBMSCs was promoted under extracellular alkali conditions (pH 7.6~8.0) via CaSR/ERK pathway. On the other hand, the differentiation was inhibited/delayed via decreased ALP activity besides gene expression at pH 8.0. CONCLUSION: Extracellular alkali or acidic surrounding according to pH alteration can play a crucial role in hBMSC behavior including the proliferation and the differentiation.

The Effects of Extracellular pH on Proliferation and Differentiation of human Bone Marrow Stem Cells

OBJECTIVES: The purpose of this study is to identify whether the change of pH affects the proliferation and the differentiation of human bone marrow stem cells (hBMSCs) and what mechanism is underlied. METHODS: To achieve objective of this study, hBMSCs were cultivated in the conditioned media adjusted to potential of hydrogen (pH) ranging from 6.4 to 8.0 using addition of hydrochloric acid (HCl) and sodium hydroxide (NaOH). The ratio of proliferation of hBMSCs according to the change of pH was measured for 24 h, 48 h, and 72 h using water-soluble tetrazolium salt (WST)-8 method. To elucidate the mechanism involved, hBMSCs was subjected to blocking extracellular signal-regulated kinases (ERK) and calcium sensing receptor (CaSR) activation. The Osteogenic-related genes and alkaline phosphatase (ALP) activity were tested under the conditioned media. RESULTS: The proliferation of hBMSCs was promoted under extracellular alkali conditions (pH 7.6~8.0) via CaSR/ERK pathway. On the other hand, the differentiation was inhibited/delayed via decreased ALP activity besides gene expression at pH 8.0. CONCLUSION: Extracellular alkali or acidic surrounding according to pH alteration can play a crucial role in hBMSC behavior including the proliferation and the differentiation.

The Effect of Cefazolin on Mechanical Properties and Antibacterial Reactions of Calcium Phosphate Cement / 대한정형외과학회잡지

PURPOSE: The purpose of this study is to evaluate the change of mechanical properties and the effect of antibacterial reactions in calcium phosphate cement (CPC) mixed with cefazolin. MATERIALS AND METHODS: We made CPC and a sodium alginate solution and we mixed in variable dosages of cefazolin and then we made a standard sized cement mold. With that we performed compression stress tests, drug releasing tests and antibacterial tests. RESULTS: We found the typical appearance of hydroxyapatite (HA) in the cement mixed with cefazolin. The compressive strength of the cement mixed with cefazolin was higher than that of the cement not mixed with cefazolin and the higher strength cement had a smaller pore size and less porosity. The sodium alginate solution showed the maximum compressive strength at 2 & 4 wt%, but this was decreased at 6 wt%. Cefazolin was released in proportion to the concentration for the first 8 days on the drug releasing test and then a similar amount was released until the tenth day. An antibacterial effect was detected at all dosages of cefazolin on the antibacterial test. CONCLUSION: The compressive strength of the cement mixed with cefazolin was higher than that of the cement not mixed with cefazolin. The drug was released from the cement in a proper fashion and the antibacterial effect was preserved.

Study about Biodegradation of Hydroxyapatite and beta-tricalcium Phosphate Coating Layer

OBJECTIVE: Biodegradation of synthetic calcium phosphate depends on two mechanisms; dissolution in body fluid and a cellular reaction. The chemical dissolution process is well understood, but details of the cellular reaction are not yet fully understood. Thus, we investigated whether the resorption mechanisms of hydroxyapatite (HA) and beta-tricalcium phosphate (beta-TCP) coating layer were identical, and, if not, what differences were observed. METHODS: Titanium discs, 12 mm in diameter and 2 mm in thickness, were coated with HA (n = 40) or beta-TCP (n = 40) by a dip and spin method. In each group, specimens were divided into two subgroups; the Dissolution group (D) and the Osteoclast Culture group (C). Discs in the D group were immersed in the distilled water or cell culture medium for 5 days, whereas, in the C group, osteoclast-like cells, which were obtained from giant cell tumor of bone, were seeded onto specimens and cultured for 5 days. The degradation characteristics of the surface coating were observed by light microscopy and scanning electron microscopy. RESULTS: Extent of the cracks and denudations were much greater in the beta-TCP coating than the HA coating after dissolution. In the C groups, the mean area fraction of resorption lacunae in the HA-C group was 11.62%, which was significantly higher than the 0.73% noted in the beta-TCP-C group (P = 0.001). CONCLUSION: The resorption mechanism of HA and beta-TCP coating was different and the beta-TCP coating was degraded principally by dissolution and separation from the implant, but the HA coating was resorbed by osteoclastic activity.

HIF-1alpha and VEGF Expression in Fracture Healing / 대한정형외과학회잡지

PURPOSE: To elucidate the relation between fracture healing and angiogenesis, we checked expression of Hypoxia-inducible factor (HIF) and Vascular endothelial growth factor (VEGF) in hypoxic cell cultures and the callus from a rat femur fracture model. MATERIALS AND METHODS: Human osteoblasts, chondrocytes, and rat ST2 cells were cultured in DME/F12 media with 10% FBS. Hypoxic DME/F12 media (PO2<60 mmHg) was generated by bubbling with 95% N2 and 5% CO2 and added to cells. After 2, 6, and 24 hours, RNA and proteins were collected for reverse transcription - polymerase chain reaction (RT-PCR) and Western blot. In addition, immunocytochemistry and siRNA treatment for HIF-1alpha were performed. Next, femurs from 9-week SD rats were fractured after fixation with needles. The rats were sacrificed at post-fracture day (PFD) 3, 5, 7, 10, 14, 21 and calluses were collected for RT-PCR and Western blot. RESULTS: HIF-1alpha and HIF-2alpha expression were not increased in RT-PCR but protein levels were increased. VEGF expression in RT-PCR was increased. Treatment with siRNA directed towards HIF inhibited VEGF expression. In the rat fracture callus, HIF-1alpha and VEGF expression peaked between PFD 5 and 7 and decreased after PFD 10. In contrast to cell culture, mRNA expression of HIF-1alpha was increased at PFD 7. CONCLUSION: HIF-1alpha and VEGF peaked early in fracture healing. With expression decreasing as O2 tension increased. Further study is needed to identify other factors affecting chondrogenic differentiation.

Influence of Associated Medical Diseases and Complications on Functional Improvement after Rehabilitation in Patients with Stroke

OBJECTIVE: To investigate the influence of associated medical diseases and complications on functional improvement after in-patient through stroke rehabilitation. METHOD: We performed a retrospective analysis on medical records of 183 stroke patients who had admitted to the department of rehabilitation medicine. Functional Independence Measure (FIM), Modified Barthel Index (MBI) at admission and discharge were used to assess the functional status. We investigated medical diseases, such as hypertension, diabetes, myocardial infarct, atrial fibrillation osteoarthritis, rheumatoid arthritis, previous history of stroke and complications such as dementia, post-stroke depression, central post-stroke pain, complex regional pain syndrome, neglect and aphasia. RESULTS: Post-stroke patients with myocardial infarct, atrial fibrillation, osteoarthritis, dementia, aphasia and neglect significantly showed lower gain of FIM and MBI, lower FIM and MBI efficacy during inpatient rehabilitation compared to without those (p0.05). Total numbers of associated medical diseases and complications negatively affect on FIM and MBI efficacy (p<0.05). CONCLUSION: Therefore, it may be important to early detect and manage associated medical diseases and complications in post-stroke patients during rehabilitation, which improve the overall functional recovery of the patients.

A Case of Ganglioneuroblastoma Presenting as Weakness of the Lower Extremities

Ganglioneuroblastoma is a rare tumor originating from the sympathetic chain. It is intermediate between the highly malignant neuroblastoma and the benign ganglioneuroma. The predilection sites of ganglioneuroblastoma are the retroperitoneum and mediastinum but involvement of the structures around the spinal cord is infrequent. A 4 year-old girl who had progressive weakness in both lower extremities had a tiptoe gait for 2 months. A magnetic resonance imaging (MRI) study revealed a heterogeneous enhancing mass in the right mediastinum and paraspinal areas. A biopsy of the mass confirmed a ganglioneuroblastoma with many neuroblasts and nodules of ganglion cells. This is an unusual case of a child with spastic paraplegia caused by ganglioneuroblastoma of the thoracic cavity.

The Effects of Anti-inflammatory Drugs on Histologic Findings of the Experimental Prolotherapy Model

OBJECTIVE: To investigate the effects of nonsteroidal anti- inflammatory drugs (NSAIDs) and acetaminophen on histologic changes of the Achilles tendon in an experimental prolotherapy model. METHOD: The right Achilles tendon of 60 rats was injected with 20% dextrose on experimental day 1, 7, and day 14, whereas the left was not injected and used as control. Rats were divided into 3 subgroups: NSAIDs medication group (10 mg/kg/day), acetaminophen medication group (100 mg/ kg/day) and no medication group. Medications were given for 3 consecutive days after each injection. Rats were sacrificed at 3 and 6 weeks after first injection. The transverse diameter of gross specimen, the number of fibroblasts on light microscope, and the distribution of collagen fibril on electron microscope were assessed. RESULTS: The transverse diameter and the count of fibroblasts of all groups increased significantly in the injected tendon compared to the non-injected tendon. However, there were no differences among all groups significantly (p<0.05). On electron micrograph, fibril diameters of injected tendon consisted of mainly smaller sizes with the intermediate sizes. CONCLUSION: Prolotherapy enhances fibroblastic stimulation and elaboration of extracellular matrix. Short term use of NSAIDs may not have any adverse effects on tissue proliferation after prolotherapy.