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The concentration of bone marrow (BM) aspirate (BMA) is increasingly valued for bone and cartilage repair, particularly with the rarity and donor-variability of BM-multipotential stromal cells (BM-MSCs). The present study aimed to assess BM-MSC yield following BM concentration using a fast and compact-sized vertical centrifugation system. BMA concentrate (BMAC) was separated in a 1 min process and collected easily after an automatic discarding of plasma and red blood cells. A significant increase in CD45low CD271high cells per BMAC volume (measured using flow-cytometry) was noted (4-fold, p = 0.0001). Additionally, the vertical centrifugation system helped to enrich colony numbers (assessed by CFU-F assays) in BMAC comparably with conventional centrifugation systems, BioCUE™ and SmartPReP-2® (4.3-fold, 4.6-fold and 3-fold, respectively). Next, a functional assessment of BM-MSCs processed by vertical centrifugation was performed, and MSC viability and proliferation were not affected. Also, these BM-MSCs showed similar alkaline phosphatase and calcium levels to those of BMA-MSCs when osteogenically induced. Furthermore, glycosaminoglycans and Nile red levels in addition to the gene expression assays confirmed that there was no significant change in chondrogenic or adipogenic abilities between BMA-MSCs and BMAC-MSCs. The expression levels of selected angiogenic and immunomodulatory mediators were also similar between the two groups. Collectively, the vertical centrifugation system helped to enrich BM-MSCs effectively, while maintaining cell viability and functions. Thus, such a vertical centrifugation system for BM concentration can be valuable for various regenerative therapies.

Barrier membranes are popularly used for guided bone regeneration (GBR). However, more knowledge is needed to assess how these membranes could be of therapeutic value when populated with native multipotent stromal cells (MSCs), particularly in the orthopaedic field. The present manuscript investigated the activities of human bone marrow-multipotent stromal cells (BM-MSCs) when loaded on to two differently structured pure collagen membranes. A crosslinked collagen membrane (CS) was tested versus a non-crosslinked bilayer collagen membrane, Bio-Gide® (BG). Following loading with BM aspirate containing native MSCs, cell attachment to the membranes was examined using electron microscopy and flow cytometry. Furthermore, alkaline phosphatase (ALP) expression and calcium deposition levels were investigated for these BM-aspirate-loaded membranes. Culture-expanded BM-MSCs were also used to load membranes and confirm the MSC functional data. All membranes supported BM-MSC attachment. However, larger numbers of attached BM-MSCs were detected for CS as compared to BG (p = 0.0010). In osteogenic medium, ALP activity was higher for CS than BG (p = 0.0312). Total calcium deposition (not normalised to cell count) was also higher for CS than BG (p = 0.0073). Consistently, the normalised secreted vascular endothelial growth factor A (VEGF-A) levels were higher in BM-MSCs loaded on CS relative to BG (p = 0.0302). Collectively, both collagen membranes supported the osteogenic functions of BM-MSCs. However, CS was found to be overall superior probably since it provided more BM-MSC attachment. These collagen membranes could potentially be used to improve GBR outcomes in orthopaedic applications.

A large proportion of ankle osteoarthritis (OA) has an early onset and is post‐traumatic. Surgical interventions have low patient satisfaction and relatively poor clinical outcome, whereas joint‐preserving treatments, which rely on endogenous multipotential stromal cells (MSCs), result in suboptimal repair. This study investigates MSC presence and potency in OA‐affected talocrural osteochondral tissue. Bone volume fraction (BV/TV) changes for the loading region trabecular volume and subchondral bone plate (SBP) thickness in OA compared with healthy tissue were investigated using microcomputed tomography. CD271‐positive MSC topography was related to bone and cartilage damage in OA tissue, and in vitro MSC potency was compared with control healthy iliac crest (IC) MSCs. A 1.3‐ to 2.5‐fold SBP thickening was found in both OA talus and tibia, whereas BV/TV changes were depth‐dependent. MSCs were abundant in OA talus and tibia, with similar colony characteristics. Tibial and talar MSCs were tripotential, but talar MSCs had 10‐fold lower adipogenesis and twofold higher chondrogenesis than IC MSCs (P =.01 for both). Cartilage damage in both OA tibia and talus correlated with SBP thickening and CD271+ MSCs was 1.4‐ to twofold more concentrated near the SBP. This work shows multipotential MSCs are present in OA talocrural subchondral bone, with their topography suggesting ongoing involvement in SBP thickening. Potentially, biomechanical stimulation could augment the chondrogenic differentiation of MSCs for joint‐preserving treatments. [ABSTRACT FROM AUTHOR]

Background aims Although intra-articular injection of platelet products is increasingly used for joint regenerative approaches, there are few data on their biological effects on joint-resident multipotential stromal cells (MSCs), which are directly exposed to the effects of these therapeutic strategies. Therefore, this study investigated the effect of platelet lysate (PL) on synovial fluid–derived MSCs (SF-MSCs), which in vivo have direct access to sites of cartilage injury. Methods SF-MSCs were obtained during knee arthroscopic procedures (N = 7). Colony forming unit–fibroblast (CFU-F), flow-cytometric phenotyping, carboxyfluorescein succinimidyl ester-based immunomodulation for T-cell and trilineage differentiation assays were performed using PL and compared with standard conditions. Results PL-enhanced SF-MSC (PL-MSC) proliferation as CFU-F colonies was 1.4-fold larger, and growing cultures had shorter population-doubling times. PL-MSCs and fetal calf serum (FCS)-MSCs had the same immunophenotype and similar immunomodulation activities. In chondrogenic and osteogenic differentiation assays, PL-MSCs produced 10% more sulfated-glycosaminoglycan (sGAG) and 45% less Ca ++ compared with FCS-MSCs, respectively. Replacing chondrogenic medium transforming growth factor-β3 with 20% or 50% PL further increased sGAG production of PL-MSCs by 69% and 95%, respectively, compared with complete chondrogenic medium. Also, Dulbecco's Modified Eagle's Medium high glucose (HG-DMEM) plus 50% PL induced more chondrogenesis compared with HG-DMEM plus 10% FCS and was comparable to complete chondrogenic medium. Conclusions This is the first study to assess SF-MSC responses to PL and provides biological support to the hypothesis that PL may be capable of modulating multiple functional aspects of joint resident MSCs with direct access to injured cartilage. [ABSTRACT FROM AUTHOR]

Bone marrow (BM) contains stem cells for both hematopoietic and nonhematopoietic lineages. Hematopoietic stem cells enable hematopoiesis to occur in a controlled manner in order to accurately compensate for the loss of short- as well as long-lived mature blood cells. The physiological role of nonhematopoietic BM stem cells, often referred to as multipotential stromal cells or skeletal stem cells (SSCs), is less understood. According to an authoritative current opinion, the main function of SSCs is to give rise to cartilage, bone, marrow fat and hematopoiesis-supportive stroma, in a specific sequence during embryonic and postnatal development. This review outlines recent advances in the understanding of origins and homeostatic functions of SSCs in vivo and highlights current and future SSC-based treatments for skeletal and joint disorders. [ABSTRACT FROM AUTHOR]

Aim: To enumerate and characterize multipotential stromal cells (MSCs) in a cellular bone allograft and compare with fresh age-matched iliac crest bone and bone marrow (BM) aspirate. Materials & methods: MSC characterization used functional assays, confocal/scanning electron microscopy and whole-genome microarrays. Resident MSCs were enumerated by flow cytometry following enzymatic extraction. Results: Allograft material contained live osteocytes and proliferative bone-lining cells defined as MSCs by phenotypic and functional capacities. Without cultivation/expansion, the allograft displayed an 'osteoinductive' molecular signature and the presence of CD45-CD271+CD73+CD90+CD105+ MSCs; with a purity over 100-fold that of iliac crest bone. In comparison with BM, MSC numbers enzymatically released from 1 g of cellular allograft were equivalent to approximately 45 ml of BM aspirate. Conclusion: Cellular allograft bone represents a unique nonimmune material rich in MSCs and osteocytes. This osteoinductive graft represents an attractive alternative to autograft bone or composite/synthetic grafts in orthopedics and broader regenerative medicine settings. [ABSTRACT FROM AUTHOR]

ABSTRACT Graft expanders are bone scaffolds used, in combination with autografts, to fill large bone defects in trauma surgery. This study investigates the graft expander potential of a natural bone substitute Orthoss® by studying its ability to support attachment, growth and osteogenic differentiation of neighboring multipotential stromal cells (MSCs). Material consisting of bone marrow (BM) aspirate and reamer-irrigator-aspirator (RIA)-harvested autograft bone was co-cultured with commercially available Orthoss® granules. Native MSCs attached to Orthoss® were expanded and phenotypically characterized. MSCs egress from neighboring cancelous bone was assessed in 3D Matrigel co-cultures. MSC differentiation was evaluated using scanning electron microscopy and measuring alkaline phosphatase (ALP) activity per cell. CD45+ hematopoietic lineage cells and highly proliferative CD90+CD73+CD105+ MSCs preferentially colonized Orthoss® granules, over RIA bone chips. MSC colonization was followed by their intrinsic osteogenic differentiation, assessed as mineral deposition and gradual rise in ALP activity, even in the absence of osteogenic stimuli. When in contact with mixed cell populations and RIA chips, Orthoss® granules support the attachment, growth and osteogenic differentiation of neighboring MSCs. Therefore, natural bone substitutes similar to Orthoss® can be used as void fillers and graft expanders for repairing large bone defects in conjunction with autologous BM aspirates and autografts. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 31:1950-1958, 2013 [ABSTRACT FROM AUTHOR]

Background Aims: Mesenchymal stromal cells (MSCs) are regenerative and immuno-privileged cells that are used for both tissue regeneration and treatment of severe inflammation-related disease. For quality control of manufactured MSC batches in regard to mature fat cell contamination, a quantitative method for measuring adipogenesis is needed. Methods: Four previously proposed methods were validated with the use of bone marrow (BM) MSCs during a 21-day in vitro assay. Oil red staining was scored semiquantitatively; peroxisome proliferator activated receptor-γ and fatty acid binding protein (FABP)4 transcripts were measured by quantitative real-time polymerase chain reaction; FABP4 protein accumulation was evaluated by flow cytometry; and Nile red/4',6-diamidino-2-phenylindole (DAPI) ratios were measured in fluorescent microplate assay. Skin fibroblasts and MSCs from fat pad, cartilage and umbilical cord were used as controls. Results: Oil red staining indicated considerable heterogeneity between BM donors and individual cells within the same culture. FABP4 transcript levels increased 100- to 5000-fold by day 21, with large donor variability observed. Flow cytometry revealed increasing intra-culture heterogeneity over time; more granular cells accumulated more FABP4 protein and Nile red fluorescence compared with less granular cells. Nile red increase in day-21 MSCs was ∼5- and 4-fold, measured by flow cytometry or microplate assay, respectively. MSC proliferation/apoptosis was accounted through the use of Nile red/DAPI ratios; adipogenesis levels in day-21 BM MSCs increased ∼13-fold, with significant correlations with oil red scoring observed for MSC from other sources. Conclusions: Flow cytometry permits the study of MSC differentiation at the single-cell level and sorting more and less mature cells from mixed cell populations. The microplate assay with the use of the Nile red/DAPI ratio provides rapid quantitative measurements and could be used as a low-cost, high-throughput method to quality-control MSC batches from different tissue sources. [ABSTRACT FROM AUTHOR]

Multipotential stromal cells or mesenchymal stem cells (MSCs) have been proposed as aids in regenerating bone and adipose tissues, as these cells form osteoblasts and adipocytes. A major obstacle to this use of MSC is the initial loss of cells postimplantation. This cell death in part is due to ubiquitous nonspecific inflammatory cytokines such as FasL generated in the implant site. Our group previously found that soluble epidermal growth factor (sEGF) promotes MSC expansion. Furthermore, tethering EGF (tEGF) onto a two-dimensional surface altered MSC responses, by restricting epidermal growth factor receptor (EGFR) to the cell surface, causing sustained activation of EGFR, and promoting survival from FasL-induced death. sEGF by causing internalization of EGFR does not support MSC survival. However, for tEGF to be useful in bone regeneration, it needs to allow for MSC differentiation into osteoblasts while also protecting emerging osteoblasts from apoptosis. tEGF did not block induced differentiation of MSCs into osteoblasts, or adipocytes, a common default MSC-differentiation pathway. MSC-derived preosteoblasts showed increased Fas levels and became more susceptible to FasL-induced death, which tEGF prevented. Differentiating adipocytes underwent a reduction in Fas expression and became resistant to FasL-induced death, with tEGF having no further survival effect. tEGF protected undifferentiated MSC from combined insults of FasL, serum deprivation, and physiologic hypoxia. Additionally, tEGF was dominant in the face of sEGF to protect MSC from FasL-induced death. Our results suggest that MSCs and differentiating osteoblasts need protective signals to survive in the inflammatory wound milieu and that tEGF can serve this function. S tem C ells 2013;31:104-116 [ABSTRACT FROM AUTHOR]

Background aims. The manufacture of multipotential stromal cell (MSC)-based products is costly; therefore, a rapid evaluation of bone marrow (BM) 'quality' with respect to MSC content is desirable. The aim of this study was to develop a rapid single-platform assay to quantify MSC in BM aspirates. Methods. Aspirated MSC were enumerated using the CD45-/low CD271bright phenotype and AccuCheck counting beads and compared with a classic colony-forming unit-fibroblast (CFU-F) assay. The phenotype of CD45-/low CD271bright cells was defined using a range of MSC (CD73, CD105, CD90) and non-MSC (CD31, CD33, CD34, CD19) markers. The effect of aspirated BM volume on MSC yield was also determined. Results. CD45-/low CD271bright cells had a classic MSC phenotype (CD73++ CD105++ CD90++ ). Their numbers correlated positively with CFU-F counted manually ( R == 0.81, P < 0.001) or using automatic measurements of surface area occupied by colonies ( R == 0.66, P < 0.001). Simultaneous enumeration of CD34 ++ cells revealed donor variability ranges compatible with standard International Society of Hematotherapy and Graft Engineering (ISHGE) protocols. Aspirating larger marrow volumes gave a significant several-fold reduction in the frequency of CFU-F and CD45-/low CD271bright cells per milliliter. Therefore aspirated MSC yields can be maximized through a standardized, low-volume harvesting technique. Conclusions. Absolute quantification of CD45-/low CD271bright cells was found to be a reliable method of predicting CFU-F yields in BM aspirates. This rapid (< 40 min) procedure could be suitable for intra-operative quality control of BM aspirates prior to volume reduction/direct injection in orthopedics. In the production of culture-expanded MSC, this assay could be used to exclude samples containing low numbers of MSC, resulting in improved consistency and quality of manufactured MSC batches. [ABSTRACT FROM AUTHOR]

Abstract: 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. This article is part of a Special Issue entitled: Interactions Between Bone, Adipose Tissue and Metabolism. [Copyright &y& Elsevier]