Yield optimisation and molecular characterisation of uncultured CD271+ mesenchymal stem cells in the Reamer Irrigator Aspirator waste bag.

Bone reconstruction requires the use of autografts from patients' iliac crest (IC); for large-volume defects bone void fillers and autologous mesenchymal stem cells (MSCs) are often added. The Reamer/Irrigator/Aspirator (RIA) device provides the means of harvesting large amounts of autograft and additionally yields a waste bag containing MSCs, which is currently discarded. The aim of this study was to enumerate and characterise native MSCs from RIA waste bag and compare them to 'gold-standard' donor-matched MSCs from IC bone marrow (BM). IC-BM from age matched trauma patients was used as control. In RIA waste bags the median MSC yield established using a colony-forming fibroblast assay was 314333 (range 5 x 104-1.4 x 106), equivalent to approximately one litre of IC-BM aspirate. CD271+ cells were present at high levels in RIA waste bags, had MSC surface phenotype (CD90+CD73+CD105+CD34>sup>-CD61-CD19-CD31-CD33-) and expressed genes associated with multipotentiality, osteogenesis, adipogenesis and angiogenic support. RIA- CD271+ MSCs were transcriptionally similar to donor-matched IC-CD271+ MSCs (76 % transcripts); with the majority of bone-related and Wnt pathway molecules being expressed at comparable levels. Lower-level expression of MCAM/CD146 and 5/13 hypoxia-related molecules was found in RIA-CD271+ MSCs, potentially reflecting their native residence in a more hypoxic environment of the endosteum and bone cortex. These data suggest that long bones contain very large numbers of MSCs, transcriptionally-similar to IC-BM MSCs; they can be procured by reaming using the RIA device and used, following concentration, as autologous and potentially allogeneic bone repair therapy.

Transcriptional profile of native CD271+ multipotential stromal cells: evidence for multiple fates, with prominent osteogenic and Wnt pathway signaling activity.

OBJECTIVE: Controversy surrounds the identity and functionality of rare bone marrow-derived multipotential stromal cells (BM-MSCs), including their differentiation capabilities, their relationship to pericytes and hematopoiesis-supporting stromal cells, and the relevance of their culture-expanded progeny in studies of skeletal biology and development of cell-based therapies. The aim of this study was to clarify the nature of candidate BM-MSCs by profiling transcripts that reflect different aspects of their putative functions in vivo. METHODS: Rare, sorted BM-derived CD45(-/low) CD271(bright) (CD271) cells were analyzed using 96-gene expression arrays focused on transcripts relevant to mesenchymal-lineage differentiation (toward bone, cartilage, fat, or muscle), hematopoietic and stromal support, and molecules critical to skeletal homeostasis. These cells were compared to matched CD45+ CD271- hematopoietic-lineage cells, culture-expanded MSCs, and skin fibroblasts. When feasible, transcription was validated using flow cytometry. RESULTS: CD271 cells had a transcriptional profile consistent with the multiple fates of in vivo MSCs, evident from the observed simultaneous expression of osteogenic, adipogenic, pericytic, and hematopoiesis-supporting genes (e.g., SP7 [osterix], FABP4 [fatty acid binding protein 4], ANGPT1 [angiopoietin 1], and CXCL12 [stromal cell-derived factor 1], respectively). Compared to culture-expanded MSCs and fibroblasts, CD271 cells exhibited greater transcriptional activity, particularly with respect to Wnt-related genes (>1,000-fold increased expression of FRZB [secreted frizzled-related protein 3] and WIF1 [Wnt inhibitory factor 1]). A number of transcripts were identified as novel markers of MSCs. CONCLUSION: The native, BM-derived in vivo MSC population is endowed with a gene signature that is compatible with multiple functions, reflecting the topographic bone niche of these cells, and their signature is significantly different from that of culture-expanded MSCs. This indicates that studies of the biologic functions of MSCs in musculoskeletal diseases, including osteoporosis and osteoarthritis, should focus on in vivo MSCs, rather than their culture-adapted progeny.

Transient Existence of Circulating Mesenchymal Stem Cells in the Deep Veins in Humans Following Long Bone Intramedullary Reaming.

The biology of mesenchymal stem cells (MSCs) in humans is incompletely understood and a possible role of systemically circulating cells in health and autoimmune disease remains controversial. Physiological movement of bone marrow MSCs to sites of injury would support the rationale for intravenous administration for relocation to damaged organs. We hypothesized that biophysical skeletal trauma rather than molecular cues may explain reported MSC circulation phenomena. Deep-femoral vein (FV) and matched peripheral vein blood samples (PVBs) were collected from patients undergoing lower-limb orthopaedic procedures during surgery (tibia using conventional sequential reaming, n = 9, femur using reamer/irrigator/aspirator (RIA), n = 15). PVBs were also taken from early (n = 15) and established (n = 12) rheumatoid arthritis (RA) patients and healthy donors (n = 12). Colony-forming unit-fibroblasts (CFU-Fs) were found in 17/36 FVBs but only 7/74 PVBs (mostly from femoral RIA); highly proliferative clonogenic cells were not generated. Only one colony was found in control/RA samples (n = 28). The rare CFU-Fs' MSC nature was confirmed by phenotypic: CD105+/CD73+/CD90+ and CD19-/CD31-/CD33-/CD34-/CD45-/CD61-, and molecular profiles with 39/80 genes (including osteo-, chondro-, adipo-genic and immaturity markers) similar across multiple MSC tissue controls, but not dermal fibroblasts. Analysis of FVB-MSCs suggested that their likely origin was bone marrow as only two differences were observed between FVB-MSCs and IC-BM-MSCs (ACVR2A, p = 0.032 and MSX1, p = 0.003). Stromal cells with the phenotype and molecular profile of MSCs were scarcely found in the circulation, supporting the hypothesis that their very rare presence is likely linked to biophysical micro-damage caused by skeletal trauma (here orthopaedic manipulation) rather than specific molecular cues to a circulatory pool of MSCs capable of repair of remote organs or tissues. These findings support the use of organ resident cells or MSCs placed in situ to repair tissues rather than systemic administration.

Cemented and cementless total hip replacement. Critical analysis and comparison of clinical and radiological results of 182 cases operated in Al Razi Hospital, Kuwait.

OBJECTIVE: In this study we present the results of a series of cemented Exeter and cementless Zweymuller implants. SUBJECT AND METHODS: Eighty-seven cemented and 95 cementless hip replacements for different hip pathologies were followed for an average period of 36 months for cementless and 60 months for cemented cases. Clinical results were calculated using the Merle d'Aubigne score. The orientation of the prosthetic components and the fixation of the cup and stem were analyzed. The clinical and radiological results were compared using statistical methods. RESULTS: In the average period of 36 months in cementless and 60 months in cemented hip replacements the clinical results improved significantly when compared with the preoperative score (p < 0.05). Sixty-seven cemented acetabular cups (77.1%) were in the desired position (30-50 degrees ) and 20 cemented cups (22.9%) were outside this range. Seventy-six cups (80%) were in the desired degree of abduction and 19 (20%) were outside this range. All cups except 1 were anteverted or neutral. Of the femoral stems, 173 were in the neutral position, 5 in the valgus and 4 in the varus position. Cemented cups were more commonly loose and cemented and cementless stems did equally well. No significant differences in rate of complications were found. CONCLUSION: Cementless acetabular implants had better clinical results and a lower loosening rate at 3 years of follow-up compared to cemented implants at 5 years of follow-up. The cemented femoral implants were equally stable compared to the cementless ones.

High abundance of CD271(+) multipotential stromal cells (MSCs) in intramedullary cavities of long bones.

Aspiration of iliac crest bone marrow (ICBM) remains the most frequent technique used in harvesting multipotential stromal cells (MSCs) for bone regeneration. Although this tissue type is easily accessed by a surgeon, it has a low frequency of MSCs, which is significant given the high cell numbers required for bone regeneration strategies. Lipoaspirates possess higher MSC frequencies, albeit cells with a differentiation profile less suited to orthopaedic interventions. Intra-medullary cavities of long bones have previously been shown to harbour MSCs in animals, however evaluation of their frequency, differentiation capacity and phenotype in humans had not previously been performed. Long bone fatty bone marrow (LBFBM) was collected prior to harvesting bone graft. Basic cellular compositions of donor-matched LBFBM and ICBM aspirates, including the numbers of CD34(+) hematopoietic stem cells and CD31(+) endothelial cells, were similar. MSCs were enumerated using colony-forming-unit-fibroblast assays and flow cytometry for the presence of a resident LBFBM CD45(-/low) CD271(+) MSC population and revealed a trend for higher MSC numbers (average 5 fold, n=6) per millilitre of LBFBM compared to donor-matched ICBM. Functional characteristics of resident MSCs, including their growth rates, differentiation potentials and surface phenotypes (CD73(+)CD105(+)CD90(+)) before and after culture-amplification, were similar. Enhanced numbers of MSCs could be recovered following brief enzymatic treatment of solid fragments of LBFBM. Our findings therefore reveal that the intramedullary cavity of the human femur is a depot of MSCs, which, although closely associated with fat, have a differentiation profile equivalent to ICBM. This anatomical site is frequently accessed by the orthopaedic/trauma surgeon and aspiration of the intramedullary cavity represents a 'low-tech' method of harvesting potentially large numbers of MSCs for regenerative therapies and research.

Radioanatomical measurements of the medullary cavity of the humerus in Kuwait: ethnic differences and clinical implications for fracture fixation.

OBJECTIVE: To compare the difference in diameter of the medullary cavity of the humerus in Arab and South Asian patients in Kuwait. SUBJECTS AND METHODS: In 46 patients (29 Arab, 17 South Asian) treated at Al-Razi Hospital, Kuwait, for diaphyseal fracture of the humerus, the medullary cavity of the humerus on the radiographs of the uninjured (contralateral side) was examined. Bone density was also measured. Statistical significance was calculated by parametric and nonparametric test using SPSS package. RESULTS: South Asians had a significantly narrower medullary cavity (p < 0.05) and higher cortical indices (p < 0.05) than Arabs, and also a narrower external diameter of the humerus at levels 5 and 6 (p < 0.05). CONCLUSION: In cases of fracture of the humerus, radiographic measurements of the diameter of the medullary cavity of the uninjured side were essential in selecting the appropriate fixation device.