Phenotypic Characterization of Bone Marrow Mononuclear Cells and Derived Stromal Cell Populations from Human Iliac Crest, Vertebral Body and Femoral Head.

(1) In vitro, bone marrow-derived stromal cells (BMSCs) demonstrate inter-donor phenotypic variability, which presents challenges for the development of regenerative therapies. Here, we investigated whether the frequency of putative BMSC sub-populations within the freshly isolated mononuclear cell fraction of bone marrow is phenotypically predictive for the in vitro derived stromal cell culture. (2) Vertebral body, iliac crest, and femoral head bone marrow were acquired from 33 patients (10 female and 23 male, age range 14-91). BMSC sub-populations were identified within freshly isolated mononuclear cell fractions based on cell-surface marker profiles. Stromal cells were expanded in monolayer on tissue culture plastic. Phenotypic assessment of in vitro derived cell cultures was performed by examining growth kinetics, chondrogenic, osteogenic, and adipogenic differentiation. (3) Gender, donor age, and anatomical site were neither predictive for the total yield nor the population doubling time of in vitro derived BMSC cultures. The abundance of freshly isolated progenitor sub-populations (CD45-CD34-CD73+, CD45-CD34-CD146+, NG2+CD146+) was not phenotypically predictive of derived stromal cell cultures in terms of growth kinetics nor plasticity. BMSCs derived from iliac crest and vertebral body bone marrow were more responsive to chondrogenic induction, forming superior cartilaginous tissue in vitro, compared to those isolated from femoral head. (4) The identification of discrete progenitor populations in bone marrow by current cell-surface marker profiling is not predictive for subsequently derived in vitro BMSC cultures. Overall, the iliac crest and the vertebral body offer a more reliable tissue source of stromal progenitor cells for cartilage repair strategies compared to femoral head.

A Perfusion Culture System for Assessing Bone Marrow Stromal Cell Differentiation on PLGA Scaffolds for Bone Repair.

Biomaterials development for bone repair is currently hindered by the lack of physiologically relevant in vitro testing systems. Here we describe the novel use of a bi-directional perfusion bioreactor to support the long term culture of human bone marrow stromal cells (BMSCs) differentiated on polylactic co-glycolic acid (PLGA). Primary human BMSCs were seeded onto porous PLGA scaffolds and cultured in static vs. perfusion culture conditions for 21 days in osteogenic vs. control media. PLGA scaffolds were osteoconductive, supporting a mature osteogenic phenotype as shown by the upregulation of Runx2 and the early osteocyte marker E11. Perfusion culture enhanced the expression of osteogenic genes Osteocalcin and Osteopontin. Extracellular matrix deposition and mineralisation were spatially regulated within PLGA scaffolds in a donor dependant manner. This, together with the observed upregulation of Collagen type X suggested an environment permissive for the study of differentiation pathways associated with both intramembranous and endochondral ossification routes of bone healing. This culture system offers a platform to assess BMSC behavior on candidate biomaterials under physiologically relevant conditions. Use of this system may improve our understanding of the environmental cues orchestrating BMSC differentiation and enable fine tuning of biomaterial design as we develop tissue-engineered strategies for bone regeneration.

A doxycycline inducible, adenoviral bone morphogenetic protein-2 gene delivery system to bone.

We report the novel use of a tuneable, non-integrating viral gene delivery system to bone that can be combined with clinically approved biomaterials in an 'off-the-shelf' manner. Specifically, a doxycycline inducible Tet-on adenoviral vector (AdTetBMP-2) in combination with mesenchymal stromal cells (MSCs), fibrin and a biphasic calcium phosphate ceramic (MBCP®) was used to repair large bone defects in nude rats. Bone morphogenetic protein-2 (BMP-2) transgene expression could be effectively tuned by modification of the doxycycline concentration. The effect of adenoviral BMP-2 gene delivery upon bone healing was investigated in vivo in 4 mm critically sized, internally fixated, femoral defects. MSCs were transduced either by direct application of AdTetBMP-2 or by pre-coating MBCP granules with the virus. Radiological assessment scores post-mortem were significantly improved upon delivery of AdTetBMP-2. In AdTetBMP-2 groups, histological analysis revealed significantly more newly formed bone at the defect site compared with controls. Newly formed bone was vascularized and fully integrated with nascent tissue and implanted biomaterial. Improvement in healing outcome was achieved using both methods of vector delivery (direct application vs. pre-coating MCBP). Adenoviral delivery of BMP-2 enhanced bone regeneration achieved by the transplantation of MSCs, fibrin and MBCP in vivo. Importantly, our in vitro and in vivo data suggest that this can be achieved with relatively low (ng/ml) levels of the growth factor. Our model and novel gene delivery system may provide a powerful standardized tool for the optimization of growth factor delivery and release for the healing of large bone defects. Copyright © 2016 John Wiley & Sons, Ltd.

Corrigendum to "Effect of Short-Term Stimulation with Interleukin-1ß and Differentiation Medium on Human Mesenchymal Stromal Cell Paracrine Activity in Coculture with Osteoblasts".

[This corrects the article DOI: 10.1155/2015/714230.].

Improving translation success of cell-based therapies in orthopaedics.

There is a clear discrepancy between the growth of cell therapy and tissue engineering research in orthopaedics over the last two decades and the number of approved clinical therapies and products available to patients. At the 2015 annual meeting of the Orthopaedic Research Society, a workshop was held to highlight important considerations from the perspectives of an academic scientist, clinical researcher, and industry representative with the aim of helping researchers to successfully translate their ideas into clinical and commercial reality. Survey data acquired from workshop participants indicated an overall positive opinion on the future potential of cell-based therapies to make a significant contribution to orthopaedic medicine. The survey also indicated an agreement on areas requiring improvement in the development of new therapies, specifically; increased support for fundamental research and education and improved transparency of regulatory processes. This perspectives article summarises the content and conclusions of the workshop and puts forward suggestions on how translational success of cell-based therapies in orthopaedics may be achieved.

An In Vitro Investigation of Platelet-Rich Plasma-Gel as a Cell and Growth Factor Delivery Vehicle for Tissue Engineering.

Platelet-rich plasma (PRP) has been used for different applications in human and veterinary medicine. Many studies have shown promising therapeutic effects of PRP; however, there are still many controversies regarding its composition, properties, and clinical efficacy. The aim of this study was to evaluate the influence of different platelet concentrations on the rheological properties and growth factor (GF) release profile of PRP-gels. In addition, the viability of incorporated bone marrow-derived human mesenchymal stem cells (MSCs) was investigated. PRP (containing 1000 × 10(3), 2000 × 10(3), and 10,000 × 10(3) platelets/µL) was prepared from human platelet concentrates. Platelet activation and gelification were achieved by addition of human thrombin. Viscoelastic properties of PRP-gels were evaluated by rheological studies. The release of GFs and inflammatory proteins was measured using a membrane-based protein array and enzyme-linked immunosorbent assay. MSC viability and proliferation in PRP-gels were assessed over 7 days by cell viability staining. Cell proliferation was examined using DNA quantification. Regardless of the platelet content, all tested PRP-gels showed effective cross-linking. A positive correlation between protein release and the platelet concentration was observed at all time points. Among the detected proteins, the chemokine CCL5 was the most abundant. The greatest release appeared within the first 4 h after gelification. MSCs could be successfully cultured in PRP-gels over 7 days, with the highest cell viability and DNA content found in PRP-gels with 1000 × 10(3) platelets/µL. The results of this study suggest that PRP-gels represent a suitable carrier for both cell and GF delivery for tissue engineering. Notably, a platelet concentration of 1000 × 10(3) platelets/µL appeared to provide the most favorable environment for MSCs. Thus, the platelet concentration is an important consideration for the clinical application of PRP-gels.

Three-dimensional culture and characterization of mononuclear cells from human bone marrow.

BACKGROUND AIMS: The diverse phenotypic changes and clinical and economic disadvantages associated with the monolayer expansion of bone marrow-derived mesenchymal stromal cells (MSCs) have focused attention on the development of one-step intraoperative cells therapies and homing strategies. The mononuclear cell fraction of bone marrow, inclusive of discrete stem cell populations, is not well characterized, and we currently lack suitable cell culture systems in which to culture and investigate the behavior of these cells. METHODS: Human bone marrow-derived mononuclear cells were cultured within fibrin for 2 weeks with or without fibroblast growth factor-2 supplementation. DNA content and cell viability of enzymatically retrieved cells were determined at days 7 and 14. Cell surface marker profiling and cell cycle analysis were performed by means of multi-color flow cytometry and a 5-ethynyl-2'-deoxyuridine incorporation assay, respectively. RESULTS: Total mononuclear cell fractions, isolated from whole human bone marrow, was successfully cultured in fibrin gels for up to 14 days under static conditions. Discrete niche cell populations including MSCs, pericytes and hematopoietic stem cells were maintained in relative quiescence for 7 days in proportions similar to that in freshly isolated cells. Colony-forming unit efficiency of enzymatically retrieved MSCs was significantly higher at day 14 compared to day 0; and in accordance with previously published works, it was fibroblast growth factor-2-dependant. CONCLUSIONS: Fibrin gels provide a simple, novel system in which to culture and study the complete fraction of bone marrow-derived mononuclear cells and may support the development of improved bone marrow cell-based therapies.

High content and high throughput screening to assess the angiogenic and neurogenic actions of mesenchymal stem cells in vitro.

Mesenchymal stem cells are universally regarded across many fields of medicine, as one of the most promising cell types for use in cell-based therapies. Although not yet fully understood, the therapeutic effects of these cells are largely attributed to the trophic actions of growth factors and cytokines present in the cell secretome. Specifically, the angiogenic and neurogenic properties of these cells make them attractive for the repair of vascularised and innervated tissues. In this study, we investigate the effect of mesenchymal stem cell conditioned media on in vitro assays of angiogenesis and nerve growth. We describe the use of two state of the art high content and high throughput cell analysis systems and compare them against manual analysis techniques. Mesenchymal stem cell secretomes stimulated angiogenesis and nerve growth in vitro in a donor dependant manner. Levels of neuroregulin, platelet-derived growth factor-AA and glial-derived neurotrophic factor, positively correlated with the observed angiogenic effects of these cells. High content and high throughput cell analysis systems such as the ones used in this study, may provide rapid screening tools to assist not only with patient selection but the identification of predictive therapeutic markers to support clinical outcome monitoring for patients treated with stem cell therapies.

Effect of Short-Term Stimulation with Interleukin-1ß and Differentiation Medium on Human Mesenchymal Stromal Cell Paracrine Activity in Coculture with Osteoblasts.

INTRODUCTION: Human mesenchymal stromal cells (hMSCs) exhibit the potential to accelerate bone healing by enhanced osteogenic differentiation. Interleukin-1ß is highly expressed during fracture healing and has been demonstrated to exert a significant impact on the differentiation behaviour of hMSCs. Here, we investigate the effect of 2-hour IL-1ß stimulation on the differentiation and paracrine activity of hMSCs in coculture with osteosarcoma cells in vitro. METHODS: hMSCs from 3 donors were incubated for 2 hours with 10 ng/mL IL-1ß and subsequently cocultured with MG63-GFP cells either in control or in differentiation medium in a transwell system for 28 days. Genetic and functional effects were investigated. RESULTS: hMSCs cultured in control medium exhibited a regulatory effect on cocultured MG63-GFP cells, resulting in upregulation of osteogenic gene expression in combination with increased ALP activity. However, while stimulated hMSCs cultured under differentiation conditions exhibit signs of osteogenic differentiation, osteogenic differentiation also caused an impaired regulatory effect on the cocultured MG63-GFP cells. CONCLUSION: Short stimulation of hMSCs has the potential to modify their long-term behaviour. In addition, undifferentiated hMSCs are able to regulate osteoblast differentiation; however, this regulatory function is lost upon osteogenic differentiation in vitro. This offers a novel approach for clinical cell therapy protocols.

Cells and secretome--towards endogenous cell re-activation for cartilage repair.

Regenerative medicine approaches to cartilage tissue repair have mainly been concerned with the implantation of a scaffold material containing monolayer expanded cells into the defect, with the aim to differentiate the cells into chondrocytes. While this may be a valid approach, the secretome of the implanted cells and its effects on the endogenous resident cells, is gaining in interest. This review aims to summarize the knowledge on the secretome of mesenchymal stem cells, including knowledge from other tissues, in order to indicate how these mechanisms may be of value in repairing articular cartilage defects. Potential therapies and their effects on the repair of articular cartilage defects will be discussed, with a focus on the transition from classical cell therapy to the implantation of cell free matrices releasing specific cytokines.

Concise review: Bone marrow-derived mesenchymal stem cells change phenotype following in vitro culture: implications for basic research and the clinic.

Mesenchymal stem cells (MSCs) are increasingly being used in tissue engineering and cell-based therapies in all fields ranging from orthopedic to cardiovascular medicine. Despite years of research and numerous clinical trials, MSC therapies are still very much in development and not considered mainstream treatments. The majority of approaches rely on an in vitro cell expansion phase in monolayer to produce large cell numbers prior to implantation. It is clear from the literature that this in vitro expansion phase causes dramatic changes in MSC phenotype which has very significant implications for the development of effective therapies. Previous reviews have sought to better characterize these cells in their native and in vitro environments, described known stem cell interactions within the bone marrow, and discussed the use of innovative culture systems aiming to model the bone marrow stem cell niche. The purpose of this review is to provide an update on our knowledge of MSCs in their native environment, focusing on bone marrow-derived MSCs. We provide a detailed description of the differences between naive cells and those that have been cultured in vitro and examine the effect of isolation and culture parameters on these phenotypic changes. We explore the concept of "one step" MSC therapy and discuss the potential cellular and clinical benefits. Finally, we describe recent work attempting to model the MSC bone marrow niche, with focus on both basic research and clinical applications and consider the challenges associated with these new generation culture systems.

Spinal motor neurite outgrowth over glial scar inhibitors is enhanced by coculture with bone marrow stromal cells.

BACKGROUND CONTEXT: Transplantation of bone marrow cells into spinal cord lesions promotes functional recovery in animal models, and recent clinical trials suggest possible recovery also in humans. The mechanisms responsible for these improvements are still unclear. PURPOSE: To characterize spinal cord motor neurite interactions with human bone marrow stromal cells (MSCs) in an in vitro model of spinal cord injury (SCI). STUDY DESIGN/SETTING: Previously, we have reported that human MSCs promote the growth of extending sensory neurites from dorsal root ganglia (DRG), in the presence of some of the molecules present in the glial scar, which are attributed with inhibiting axonal regeneration after SCI. We have adapted and optimized this system replacing the DRG with a spinal cord culture to produce a central nervous system (CNS) model, which is more relevant to the SCI situation. METHODS: We have developed and characterized a novel spinal cord culture system. Human MSCs were cocultured with spinal motor neurites in substrate choice assays containing glial scar-associated inhibitors of nerve growth. In separate experiments, MSC-conditioned media were analyzed and added to spinal motor neurites in substrate choice assays. RESULTS: As has been reported previously with DRG, substrate-bound neurocan and Nogo-A repelled spinal neuronal adhesion and neurite outgrowth, but these inhibitory effects were abrogated in MSC/spinal cord cocultures. However, unlike DRG, spinal neuronal bodies and neurites showed no inhibition to substrates of myelin-associated glycoprotein. In addition, the MSC secretome contained numerous neurotrophic factors that stimulated spinal neurite outgrowth, but these were not sufficient stimuli to promote spinal neurite extension over inhibitory concentrations of neurocan or Nogo-A. CONCLUSIONS: These findings provide novel insight into how MSC transplantation may promote regeneration and functional recovery in animal models of SCI and in the clinic, especially in the chronic situation in which glial scars (and associated neural inhibitors) are well established. In addition, we have confirmed that this CNS model predominantly comprises motor neurons via immunocytochemical characterization. We hope that this model may be used in future research to test various other potential interventions for spinal injury or disease states.

Bone marrow-derived mesenchymal stem cells become antiangiogenic when chondrogenically or osteogenically differentiated: implications for bone and cartilage tissue engineering.

Osteochondral tissue repair requires formation of vascularized bone and avascular cartilage. Mesenchymal stem cells stimulate angiogenesis both in vitro and in vivo but it is not known if these proangiogenic properties change as a result of chondrogenic or osteogenic differentiation. We investigated the angiogenic/antiangiogenic properties of equine bone marrow-derived mesenchymal stem cells (eBMSCs) before and after differentiation in vitro. Conditioned media from chondrogenic and osteogenic cell pellets and undifferentiated cells was applied to endothelial tube formation assays using Matrigel™. Additionally, the cell secretome was analysed using LC-MS/MS mass spectrometry and screened for angiogenesis and neurogenesis-related factors using protein arrays. Endothelial tube-like formation was supported by conditioned media from undifferentiated eBMSCs. Conversely, chondrogenic and osteogenic conditioned media was antiangiogenic as shown by significantly decreased length of endothelial tube-like structures and degree of branching compared to controls. Undifferentiated cells produced higher levels of angiogenesis-related proteins compared to chondrogenic and osteogenic pellets. In summary, eBMSCs produce an array of angiogenesis-related proteins and support angiogenesis in vitro via a paracrine mechanism. However, when these cells are differentiated chondrogenically or osteogenically, they produce a soluble factor(s) that inhibits angiogenesis. With respect to osteochondral tissue engineering, this may be beneficial for avascular articular cartilage formation but unfavourable for bone formation where a vascularized tissue is desired.

The comparison of equine articular cartilage progenitor cells and bone marrow-derived stromal cells as potential cell sources for cartilage repair in the horse.

A chondrocyte progenitor population isolated from the surface zone of articular cartilage presents a promising cell source for cell-based cartilage repair. In this study, equine articular cartilage progenitor cells (ACPCs) and equine bone marrow-derived stromal cells (BMSCs) were compared as potential cell sources for repair. Clonally derived BMSCs and ACPCs demonstrated expression of the cell fate selector gene, Notch-1, and the putative stem cell markers STRO-1, CD90 and CD166. Chondrogenic induction revealed positive labelling for collagen type II and aggrecan. Collagen type X was not detected in ACPC pellets but was observed in all BMSC pellets. In addition, it was observed that BMSCs labelled for Runx2 and matrilin-1 antibodies, whereas ACPC labelling was significantly less or absent. For both cell types, osteogenic induction revealed positive von Kossa staining in addition to positive labelling for osteocalcin. Adipogenic induction revealed a positive result via oil red O staining in both cell types. ACPCs and BMSCs have demonstrated functional equivalence in their multipotent differentiation capacity. Chondrogenic induction of BMSCs resulted in a hypertrophic cartilage (endochondral) phenotype, which can limit cartilage repair as the tissue can undergo mineralisation. ACPCs may therefore be considered superior to BMSCs in producing cartilage capable of functional repair.

Articular cartilage glycosaminoglycans inhibit the adhesion of endothelial cells.

Articular cartilage undergoes severe loss of proteoglycan and its constituent glycosaminoglycans (GAGs) in osteoarthritis. We hypothesize that the low GAG content of osteoarthritic cartilage renders the tissue susceptible to pathological vascularization. This was investigated using an in vitro angiogenesis model assessing endothelial cell adhesion to GAG-depleted cartilage explants. Bovine cartilage explants were treated with hyaluronidase to deplete GAG content and then seeded with fluorescently tagged human endothelial cells (HMEC-1). HMEC-1 adherence was assessed after 4 hr and 7 days. The effect of hyaluronidase treatment on GAG content, chondrocyte viability, and biochemical composition of the extracellular matrix was also determined. Hyaluronidase treatment reduced the GAG content of cartilage explants by 78 ± 3% compared with that of controls (p < 0.0001). GAG depletion was associated with significantly more HMEC-1 adherence on both the surface (superficial zone) and the underside (deep zone) of the explants (both p < 0.0001). The latter provided a more favorable environment for extended culture of HMEC-1 compared with the articulating surface. Hyaluronidase treatment altered the immunostaining for chondroitin sulfate epitopes, but not for lubricin. Our results support the hypothesis that articular cartilage GAGs are antiadhesive to endothelial cells and suggest that chondroitin sulfate and/or hyaluronan are responsible. The loss of these GAGs in osteoarthritis may allow osteochondral angiogenesis resulting in disease progression.