Mohammed M. Abbas

Derivation and differentiation of bone marrow mesenchymal stem cells from osteoarthritis patients

Osteoarthritis (OA) of the knee is a degenerative joint disease caused by the progressive reduction of the articular cartilage surface that leads to reduced joint function. Cartilage degeneration occurs through gradual loss in extracellular matrix components including type II collagen and proteoglycan. Due to limited inherent self repair capacity of the cartilage, the use of cell-based therapies for articular cartilage regeneration is considered promising. Bone marrow mesenchymal stem cells (BM-MSCs) are multipotent cells and are highly capable of multilineage differentiation which render them valuable for regenerative medicine. In this study, BM-MSCs were isolated from OA patients and were characterized for MSC specific CD surface marker antigens using flowcytometry and their differentiation potential into adipocytes, osteocytes and chondrocytes were evaluated using histological and gene expression studies. BM-MSCs isolated from OA patients showed short spindle shaped morphology in culture and expressed positive MSC related CD markers. They also demonstrated positive staining with oil red O, alizarin red and alcian blue following differentiation into adipocytes, osteocytes and chondrocytes, respectively. In addition, chodrogenic related genes such as collagen type II alpha1, cartilage oligomeric matrix protein, fibromodulin, and SOX9 as well as osteocytic related genes such as alkaline phosphatase, core-binding factor alpha 1, osteopontin and RUNX2 runt-related transcription factor 2 were upregulated following chondrogenic and osteogenic differentiation respectively. We have successfully isolated and characterized BM-MSCs from OA patients. Although BM-MSCs has been widely studied and their potential in regenerative medicine is reported, the present study is the first report in our series of experiments on the BMSCs isolated from OA patients at King Abdulaziz University Hospital, Jeddah, Saudi Arabia.

Pelleted bone marrow derived mesenchymal stem cells are better protected from the deleterious effects of arthroscopic heat shock

Introduction: The impact of arthroscopic temperature on joint tissues is poorly understood and it is not known how mesenchymal stem cells (MSCs) respond to the effects of heat generated by the device during the process of arthroscopy assisted experimental cell-based therapy. In the present study, we isolated and phenotypically characterized human bone marrow mesenchymal stem cells (hBMMSCs) from osteoarthritis (OA) patients, and evaluated the effect of arthroscopic heat on cells in suspension and pellet cultures. Methods: Primary cultures of hBMMSCs were isolated from bone marrow aspirates of OA patients and cultured using DMEM supplemented with 10% FBS and characterized for their stemness. hBMMSCs (1 × 106 cells) cultured as single cell suspensions or cell pellets were exposed to an illuminated arthroscope for 10, 20, or 30 min. This was followed by analysis of cellular proliferation and heat shock related gene expression. Results: hBMMSCs were viable and exhibited population doubling, short spindle morphology, MSC related CD surface markers expression and tri-lineage differentiation into adipocytes, chondrocytes and osteoblasts. Chondrogenic and osteogenic differentiation increased collagen production and alkaline phosphatase activity. Exposure of hBMMSCs to an illuminated arthroscope for 10, 20, or 30 min for 72 h decreased metabolic activity of the cells in suspensions (63.27% at 30 min) and increased metabolic activity in cell pellets (62.86% at 10 min and 68.57% at 20 min). hBMMSCs exposed to 37, 45, and 55°C for 120 s demonstrated significant upregulation of BAX, P53, Cyclin A2, Cyclin E1, TNF-α, and HSP70 in cell suspensions compared to cell pellets. Conclusions: hBMMSC cell pellets are better protected from temperature alterations compared to cell suspensions. Transplantation of hBMMSCs as pellets rather than as cell suspensions to the cartilage defect site would therefore support their viability and may aid enhanced cartilage regeneration.

Identification of novel genetic variations affecting osteoarthritis patients

BackgroundOsteoarthritis (OA) is a progressive joint disease characterized by gradual degradation of extracellular matrix (ECM) components in the cartilage and bone. The ECM of cartilage is a highly specified structure that is mainly composed of type II collagen and provides tensile strength to the tissue via aggrecan and proteoglycans. However, changes in the ECM composition and structure can lead to loss of collagen type II and network integrity. Several risk factors have been correlated with OA including age, genetic predisposition, hereditary factors, obesity, mechanical injuries, and joint trauma. Certain genetic association studies have identified several genes associated with OA using genome-wide association studies (GWASs).ResultsWe identified several novel genetic variants affecting genes that function in several candidate causative pathways including immune responses, inflammatory and cartilage degradation such as SELP, SPN, and COL6A6.ConclusionsThe approach of whole-exome sequencing can be a promising method to identify genetic mutations that can influence the OA disease.

Relationship between human leukocyte antigen DRB1 and psoriasis in Iraqi patients

Objectives: To investigate the role of Human leukocyte antigen (HLA) Class II DRB1 in susceptibility to psoriasis in Iraqi patients. Methods: A cross-sectional comparative study including 40 patients with psoriasis attending the Department of Dermatology, Al-Kindy teaching hospital in Baghdad, Iraq, between September 2013-2015. Patient selection was carried out by the dermatologists. Ninety healthy individuals were included in the study. Human leukocyte antigen genotyping was carried out by the sequence specific oligonucleotide (SSO) method using Auto LiPA 48 (Innogenetics, Belgium) in the HLA typing research unit of the Al-Kindy College of Medicine, Baghdad, Iraq. Results: There is an increased frequency of HLA-DRB1*0102, *0306 in psoriatic patients with psoriasis p-values=0.001, confidence interval(CI)=2.492-37.487 and odds ratio(OR)=9.666. Human leukocyte antigen-DRB1*1101 is significantly associated with plaque-type psoriasis with p=0.0434, CI=1.04-1.69, and OR=4.2. Human leukocyte antigen-DRB1*0102 is significantly associated with other types of psoriasis with p=0.018, CI=0.0223-0.701, and OR=0.125. Conclusion: Human leukocyte antigen-DRB1*0102 and *0306 are significantly associated with psoriasis. Human leukocyte antigen-DRB1*1101 is significantly associated with plaque-type psoriasis. Human leukocyte antigen-DRB1*0102 is significantly associated with other types of psoriasis rather than plaque-type psoriasis.

Evaluation of in vitro chondrocytic differentiation: A stem cell research initiative at the King Abdulaziz University, Kingdom of Saudi Arabia

Mesenchymal stem cells (MSCs) from various sources have been used in cartilage differentiation with variable success. Therefore, it is of interest to evaluate the in vitro differentiation potential of the hWJSCs derived from the human umbilical cords into chondrocytes at the stem cell research facility at the King Abdulaziz University. hWJSCs are an attractive choice for tissue engineering and regenerative medical applications including cartilage regeneration. We evaluated the hWJSCs using classical histological and cartilage related gene expression studies. Some of the known parameters were re-examined for consistency at the current laboratory conditions. Early passages (P1-P4) showed short fibroblastic morphology and high expression of MSC related surface markers namely CD29 (99.9%), CD44 (97.8%), CD73 (99.6%), CD90 (95.1%) and CD105 (98.9%). MTT assay showed time dependent increase in hWJSCs proliferation by 61.06% and 206.31% at 48h and 72h respectively. Toluidine blue histology showed that hWJSCs were successfully differentiated into chondrocytes in chondrocytic differentiation medium for 21 days. Differentiated hWJSCs also showed significantly increased expression of collagen type II, aggrecan and SOX9 compared to the undifferentiated control. It should be noted that the determination of the average cell yield, the population doubling time and histological staining wtih alcian blue and/or safronin O is required in future studies for improved evaluation of differentiation. Painless derivation, abundance of stem cells that are hypo-immunogenic and safety issues makes this method advantages to MSCs derived from other sources.

Stem Cells in Bone and Articular Cartilage Tissue Regeneration

Multiple factors including trauma, infection, ageing, obesity and tumours result in bone and cartilage defects. The regeneration and functional restoration of bone and cartilage remains a significant clinical challenge. ‘Autologous grafts’ continue to remain the ‘gold standard’ in both bone and cartilage regeneration but stem cell-based therapies offer great promise in both these areas. Despite the plethora of stem cells that exist within the human body, the challenge remains in identifying the most beneficial cell type, assessing their availability, expansion under cGMP culture conditions, differentiation potential and functional restoration capacity. Embryonic stem cells; mesenchymal stem cells from the bone marrow, synovial fluid, adipose tissue and umbilical cord; and primary articular chondrocytes are some of the candidate cell types that are extensively studied in the context of bone (and cartilage) regeneration. The limited regeneration potential of cartilage adds further complexity to cartilage tissue engineering compared to the bone. However, major bone reconstruction as in the case of large bone defects due to tumour resection, fractures, and skeletal deformities is equally challenging. Incorporation of novel biomaterials, understanding the optimal cell-scaffold interactions, the addition of growth factors and provision of molecular cues are all essential in achieving effective tissue regeneration. Intensive effects in tissue regeneration can actually predispose to tissue hypertrophy, which also limits functional capacity. The current state of-the-art in both bone and cartilage regeneration is reviewed in this chapter, which highlights the importance of combined approaches involving stem/progenitor cells, biomolecules and/or biomaterials for therapies as well as rehabilitation and improvement in quality of life.

AB0102 Cartilage Paste Impregnated with Mesenchymal Stem Cells (MSCS) Repair Focal Articular Cartilage Defects in Rabbits

Background Chronic arthritis is a widespread disease1. Several therapies have been attempted to treat focal cartilage defects, but none of them resulted in full repair with formation of typical hyaline cartilage. The creation of an effective therapeutic modality is essential to minimize the side effects of conventional therapy2. Moreover, stem cell therapy may establish a prospective therapy for OA in the future. There is a requirement for additional new effective therapy opportunities3. Objectives The aim of this study is to determine the efficacy of MSC as a potential treatment for OA. Methods Focal cartilage defect was created in the medial femoral condyle of the right knee of all rabbit groups. In one group the defect was left untreated. Human umbilical cord MSCs were infused in the defect, in a second group. A third group received MSCs on a fibrin glue scaffold. A fourth group received minced cartilage impregnated with MSCs. Healing was assessed clinically, radiologically and pathologically in post-mortem samples. Results The surgical technique proved to be safe. No infection was reported and the animals were able to move normally after 8 weeks. Ex-vivo examination of the knee joints showed better healing of the defect in rabbits that received cartilage paste compared to those receiving MSCs+ fibrin glue followed by those receiving MSCs only. MRI showed persistent osteochondral defect in rabbits treated with MSCs, without and with fibrin glue, mean MOCART score was 10 points. However. The defect was smaller with fibrin glue. Complete defect fill, intact cartilage surface, and complete integration with adjacent cartilage was observed in rabbits treated with MSCs and Cartilage, mean MOCART score was 85 points. Pathologic examination revealed that MSCs, alone, induced chondrocyte proliferation and stimulated cartilage repair. A better result was obtained with MSCs + fibrin glue. The best repair was obtained when autologous cartilage paste was used. This is in agreement with MRI findings. Conclusions Repair of focal articular cartilage defects using autologous cartilage paste impregnated with MSCs appears to be successful as proven clinically, radiologically, as well as pathologically. References Neogi, T. The Epidemiology and Impact of Pain in Osteoartheritis. Osteroarthritis Cartilage. 2013 Sep; 21 (9): 1145-1153. Orth, P. Rey-Rico, A. Venkatesan, J. Madry, H. Cucchiarini, M. Current perspectives in stem cell research for knee cartilage repair. Stem Cells and Cloning: Advances and Applications. 2014:7; 1–17 Singh, J,. Stem cells and other innovative intra-articular therapies for osteoarthritis: what does the future hold?. Singh BMC Medicine 2012. 1741-7015/10/44. Acknowledgements This project was supported by Salim Bin Mahfouz chair - Stem Cells Treatment for Osteoarthritis. King Abdulaziz University- Jeedah, Saudi Arabia. Disclosure of Interest None declared

Method of delivery of bone marrow stem cells to the articular joint influences their survival during arthroscopy

Background Cartilage poor capacity to regenerate can eventually lead to osteoarthritis. We aim to restore cartilage regeneration by introducing autologus bone marrow MSCs (BMMSCs) into the damaged joint using arthroscopy. The arthroscopic procedure involves variations in temperature either to supraphysiologic or subphysiologic levels following low-flow irrigation or cryotherapy respectively [1,2]. The aim of this study was to assess whether such temperature fluctuations would influence the viability and function of delivered BMMSCs and hence the outcome of the arthroscopic procedure.