Induction of Chondrogenic Differentiation in Human Mesenchymal Stem Cells Cultured on Human Demineralized Bone Matrix Scaffold under Hydrostatic Pressure

BACKGROUND: Articular cartilage damage is still a troublesome problem. Hence, several researches have been performed for cartilage repair. The aim of this study was to evaluate the chondrogenicity of demineralized bone matrix (DBM) scaffolds under cyclic hydrostatic pressure (CHP) in vitro. METHODS: In this study, CHP was applied to human bone marrow mesenchymal stem cells (hBMSCs) seeded on DBM scaffolds at a pressure of 5 MPa with a frequency of 0.5 Hz and 4 h per day for 1 week. Changes in chondrogenic and osteogenic gene expressions were analyzed by quantifying mRNA signal level of Sox9, collagen type I, collagen type II, aggrecan (ACAN), Osteocalcin, and Runx2. Histological analysis was carried out by hematoxylin and eosin, and Alcian blue staining. Moreover, DMMB and immunofluorescence staining were used for glycosaminoglycan (GAG) and collagen type II detection, respectively. RESULTS: Real-time PCR demonstrated that applying CHP to hBMSCs in DBM scaffolds increased mRNA levels by 1.3-fold, 1.2-fold, and 1.7-fold (p < 0.005) for Sox9, Col2, and ACAN, respectively by day 21, whereas it decreased mRNA levels by 0.7-fold and 0.8-fold (p < 0.05) for Runx2 and osteocalcin, respectively. Additionally, in the presence of TGF-β1 growth factor (10 ng/ml), CHP further increased mRNA levels for the mentioned genes (Sox9, Col2, and ACAN) by 1.4-fold, 1.3-fold and 2.5-fold (p < 0.005), respectively. Furthermore, in histological assessment, it was observed that the extracellular matrix contained GAG and type II collagen in scaffolds under CHP and CHP with TGF-β1, respectively. CONCLUSION: The osteo-inductive DBM scaffolds showed chondrogenic characteristics under hydrostatic pressure. Our study can be a fundamental study for the use of DBM in articular cartilage defects in vivo and lead to production of novel scaffolds with two different characteristics to regenerate both bone and cartilage simultaneously.

Is Level of injury a determinant of quality of life among individuals with spinal cord injury? a tertiary rehabilitation center report

Objectives: The role of injury-related variables in determining health-related quality of life [HRQOL] among Iranian persons with spinal cord injury [SCI] has not yet been fully described. In this study, we compared HRQOL between individuals with injury at cervical level and those with injury at thoracolumbar sections and evaluated the discriminating value of injury level as a determinant of HRQOL among Iranian people with SCI

Methods: Individuals with SCI, who were referred to Brain and Spinal Cord Injury Research Center, were invited to participate in this investigation. HRQOL was assessed using the Short Form [SF-36] questionnaire to determine the quality of life [QOL] in eight domains: physical functioning [PF], role limitation due to physical problems [RP], bodily pain [BP], general health [GH], vitality [VT], social functioning [SF], role limitation due to emotional problems [RE], and mental health [MH]

Results: Ninety patients with paraplegia and 94 quadriplegic patients participated in this investigation. The mean score of PF domain was significantly lower in patients with injury at cervical level [p < 0.0001]. There was no significant difference in other domains of SF-36 between subjects with paraplegia and quadriplegia [p = 0.670, 0.700, 0.910, 0.710, 0.730, 0.290 and 0.850 for RP, RE, VT, MH, SF, BP and GH, respectively]. Similarly, the mean physical component summary [PCS] score was significantly higher among individuals with injury at thoracolumbar sections [p < 0.0001]. The mean mental component summary [MCS] score did not differ between the two groups [p = 0.720]

Conclusions: Patients with SCI at the cervical level have similar mental health compared to those with injury at thoracolumbar sections, which shows proper mental adaptability in quadriplegic individuals. Injury level can be used as a major determinant of the physical component of QOL among people with SCI

[Isolation, amplification and identification of mesenchymal stem cells derived from human adipose tissue]

Currently, autologous and allogeneic adipose tissues represent a ubiquitous source of material for fat reconstructive therapies. However, these approaches are limited, and often accompanied by a 40-60% reduction in graft volume following transplantation, limited proliferative capacity of mature adipocytes for ex vivo expansion, and extensive adipocyte damage encountered when harvested with conventional liposuction techniques. Recently, cell-based approaches utilizing adipogenic progenitor cells for fat tissue engineering have been developed and were reported to promote both short-term in vivo adipogenesis and to repair defect sites. The aim of this study was to isolate stem cells from fat tissue than examine the growth of stem cells by invitro tests. For human adipose stem cell isolation [hASC], subcutaneous adipose tissue sites were obtained from female subjects undergoing elective procedures. Tissues were washed 3-4 times in phosphate buffered saline [PBS] and suspended in an equal volume of PBS supplemented with 1% FCS and 0.1% collagenase type I. The tissue was placed in an agitated water bath at 37 1C. The supernatant containing mature adipocytes, was aspirated. Portions of the SVF were suspended in DMEM medium. hASCs were selected based on their ability to adhere to tissue culture plastic and subsequently expanded to 75-90% confluence. Adipose stem cells were isolated and cultured on DMEM. To assess mesenchymal origin of stem cells we used flow-cytomery technique as well as differentiation to osteocyte and chondrocyte lines. The nature of the mesenchymal cells was confirmed by flow -cytometry techniques, based on the expression of CD90, CD105, CD166, and lack of expression of hematopoietic markers of CD34, CD31, and CD45. The successful differentiation of our stem cells to osteocyte, chondrocyte had been showed by specific Alizarin-Red and Toluidine-blue staining of cells. Although we have not the results of in vivo tests to support in vivo adipogenesis either alone or in combination with natural or synthetic matrix, the results showed that stem cells isolation from adipose tissue was successful, and we provided an environment for differentiation of stem cells

EEG abnormalities in clinically diagnosed brain death organ donors in Iranian tissue bank

Brain death is defined as the permanent, irreversible and concurrent loss of all brain and brain stem functions. Brain death diagnosis is based on clinical criteria and it is not routine to use paraclinical studies. In some countries, electroencephalogram [EEG] is performed in all patients for the determination of brain death while there is some skepticism in relying on EEG as a confirmatory test for brain death diagnosis. In this study, we assessed the validity of EEG and its abnormalities in brain death diagnosis. In this retrospective study, we used 153 EEGs from medical records of 89 brain death patients in organ procurement unit of the Iranian Tissue Bank admitted during 2002-2008. We extracted and analyzed information including EEGs, which were examined by a neurologist for waves, artifacts and EEG abnormalities. The mean age of the patients was 27.2 +/- 12.7 years. The most common cause of brain death was multiple traumas due to accident [65%]. The most prevalent artifact was electrical transformer. 125 EEGs [82%] were isoelectric [ECS] and seven EEGs [5%] were depictive of some cerebral activity which upon repeat EEGs, they showed ECS patterns too. There was no relationship between cause of brain death and cerebral activity in EEGs of the patients. In this study, we could confirm ECS patterns in all brain death patients whose status had earlier been diagnosed clinically. Considering the results of this study, it seems sensible to perform EEG as a final confirmatory test as an assurance to the patients' families.