Osteoblastic differentiation of human mesenchymal ...

Biomaterials 31 (2010) 270–278

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Osteoblastic differentiation of human mesenchymal stem cells with platelet lysate Nathalie Chevallier a, b, *, Fani Anagnostou c,1, Sebastien Zilber a, d,1, Gwellaouen Bodivit a, b, Sophie Maurin a, b, Aurelie Barrault a, b, Philippe Bierling a, b, Philippe Hernigou a, d, Pierre Layrolle e, Helene Rouard a, b a

EA3952, Cellular and Tissular Bioengineering Laboratory, Henri Mondor Hospital, Paris-EST University, France Cell Therapy Facility, EFS Ile de France, 94010 Creteil, France c Laboratory for Osteoarticular Engineering, U.M.R.-C.N.R.S. 7052, and Department of Periodontology, Pitie´ Salpetrie`re Hospital, AP-HP, Paris 7-Denis Diderot University, U.F.R. of Odontology, Paris, France d Orthopaedic Surgery Department, Henri Mondor AP-HP Hospital, France e Inserm, U791, Laboratory for Osteoarticular and Dental Tissue Engineering, Faculty of Dental Surgery, University of Nantes, 1 Place Alexis Ricordeau, 44042 Nantes cedex 1, France b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 July 2009 Accepted 11 September 2009 Available online 26 September 2009

Culture of expanded mesenchymal stem cells (MSCs) seeded on biomaterials may represent a clinical alternative to autologous bone graft in bone regeneration. Foetal bovine serum (FBS) is currently used for MSC expansion, despite risks of infectious disease transmission and immunological reaction due to its xenogenic origin. This study aimed to compare the osteogenic capacities of clinical-grade human MSCs cultured with FBS or allogenic human platelet lysate (PL). In vitro, MSCs cultured in PL both accelerate the expansion rate over serial passages and spontaneously induce osteoblastic gene expression such as alkaline phosphatase (ALP), bone sialoprotein (BSP), osteopontin (Op) and bone morphogenetic protein-2 (BMP-2). In vivo, ectopic bone formation is only observed on ceramics seeded with MSCs grown in PL medium implanted under the skin of immunodeficient mice for 7 weeks. In conclusion, allogenic human PL accelerates MSC proliferation and enhances MSC osteogenic differentiation. Ó 2009 Elsevier Ltd. All rights reserved.

Keywords: Mesenchymal stem cells Platelet lysate Osteoblastic gene expression HA/bTCP scaffold

1. Introduction Large bone defects in humans are commonly treated by autologous bone grafting. This invasive technique, however, is associated with potential complications including chronic pain and risk of infection, and is limited by the amount of bone available. Alternative strategies using scaffolds coated with autologous osteogenic progenitor cells have been sought. Mesenchymal stromal cells (MSCs) are characterized by their multipotential capacity for differentiation into bone, fat, and cartilage cells [1,2]. It is also now clear that MSCs can differentiate into other tissues including tendon, muscle, neural, liver, kidney, skin and cardiac cell lineages [3,4]. These properties make MSCs attractive for regenerative medicine and in particular for replacing standard bone autograft to repair large bone defects [5–7].

* Corresponding author. EA3952, Cellular and Tissular Bioengineering Laboratory, Henri Mondor Hospital, Paris-EST University, France. Tel.: þ33 1 56 72 76 83; fax: þ33 1 56 72 76 01. E-mail address: [email protected] (N. Chevallier). 1 These authors contributed equally to this work. 0142-9612/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2009.09.043

Several animal models have shown that scaffolds used alone led to poor bone formation in vivo, as compared to biomaterials associated with MSCs [8]. Unfractionated bone marrow contains few MSCs (about 0.001%), but an amplification procedure generates MSCs on a large-scale. These amplified cells are well characterized and standardized for clinical applications. To reduce culture time between MSC isolation from bone marrow and in vitro expansion, standard culture conditions in a-MEM medium and 10% FBS need to be optimised. First, an alternative medium enriched in synthetic factors or anti-oxidative molecules may improve in vitro amplification, as suggested by Meuleman et al. [9]. Also, for clinical-scale MSC expansion, platelet lysate (PL) could be used in place of FBS which may transmit pathogens or lead to xenogeneic immunoreactions. Platelet granules contain many growth factors, including platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), transforming growth factor (TGFb), platelet factor 4 (PF-4), and platelet-derived epidermal growth factor (PDEGF) [10–12]. These growth factors, which are released from platelet lysate, have been shown to enhance MSC expansion in vitro [13,14]. Several previous studies used thrombin-activated platelets to create a platelet gel. Local platelet gel delivery is currently used for

N. Chevallier et al. / Biomaterials 31 (2010) 270–278

periodontal defects and maxillofacial reconstructive surgery [15,16]. Osteogenic induction may be due to a direct effect of the platelet gel on local osteoprogenitor recruitment, or to an indirect improvement in soft tissue wound healing. When co-administered with MSCs, platelet gel improves bone autograft or allograft integration in large animal models such as sheep or dogs [17,18], and promotes bone callus formation in humans [5]. However, other studies have reported that osteogenic differentiation of bone marrow cells loaded onto biomaterial carriers was inhibited in vitro when exposed to platelet gel [19,20]. In vivo, scaffold seeded with MSC cultured in platelet gel or scaffold seeded with platelet gel only did not induce bone formation [21,22]. Therefore, the role of platelet lysate and gel in MSC differentiation remains controversial. The in vitro MSC culture conditions may influence the gene expression profile. However, few studies have investigated how PL influences osteoblastic gene expression. In human osteosarcoma cell lines (HOS), Kanno et al. showed a slight increase in Runx2 mRNA when cells were cultured with PL in confluent conditions, although kinetics for the mRNA variations are not available [23]. Furthermore, as HOS are mature osteoblast cell lines, it would be interesting to understand osteoblastic gene expression during MSC isolation, expansion, and differentiation. In vitro, MSCs undergo mainly osteogenic differentiation through a well-defined pathway. Furthermore, the presence of glucocorticoids, ascorbic acid (AA), b-glycerophosphate (bGly) or other factors such as bone morphogenetic proteins (BMP) enhances osteoblastic differentiation [24,25]. Dexamethasone increases ALP activity and BMP6 gene expression, both markers related to osteogenic lineage differentiation [26]. However, the continuous presence of differentiation reagents, like dexamethasone, could induce apoptosis in some of the MSC population [26]. Moreover, in a rat MSC cell line model (ROB C26 cells), dexamethasone upregulates the transcription factors C/EBP and PPARg, which are specific to the adipocyte pathway, and inhibited terminal osteoblast differentiation in vitro [27]. Therefore, the addition of exogenous differentiation agents to a culture system may be useful for cell therapy in humans, but needs to be carefully evaluated. In this study, we compared the osteogenic differentiation capacity of human MSCs cultured in the absence of exogenous agents in either standard growth medium consisting of a-MEM or in a new medium (LP02) enriched in anti-oxidant complemented with either FBS or PL. The effect of culture conditions was tested both in vitro, through the study of expansion, phenotype, differentiation capacity and gene expression of human MSCs, and in vivo, by evaluating bone-forming capacity in a SCID mouse model. 2. Materials and methods 2.1. Platelet lysate (PL) preparation PL was obtained from four different platelet apheresis collections performed at the ‘‘Etablissement Francais du Sang’’ (Rungis, France). All apheresis products were biologically qualified according to the French legislation. The platelet count in each product was measured automatically (with a ABX penta 60 Cþ, Horiba ABX, Montpellier, France). Only samples containing 1 109 or more platelets/ml were retained; they were frozen at 80 C and subsequently used to obtain PL containing platelet-released growth factors. Remaining platelet bodies were eliminated by centrifugation (1400g) [14]. These PL preparations were then used in culture as FBS substitutes. 2.2. Bone marrow cell cultures Human bone marrow (3- to 5-ml volumes) was obtained from iliac crest marrow aspirates of patients undergoing standard bone marrow transplantation procedures (Henri Mondor Hospital, AP-HP Cre´teil, France) after receiving their informed consent. Bone marrow (BM) aspirates from 10 healthy donors (36–54 years old) were used. Nucleated cells from fresh marrow were seeded at 2 105/cm2 in 225-cm2 flasks. MSCs were expanded in a-modified Eagle’s medium (aMEM) (Invitrogen, Cergy Pontoise, France) or LP02 medium (MacoPharma, Tourcoing, France). LP02 is

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a similar culture product such as a-MEM plus anti-oxidant molecules. MSCs were expanded in aMEM or LP02 containing either 10% foetal bovine serum (FBS) (StemCell Technologies, Grenoble, France) or 5% PL [14]. Each medium was supplemented with 0.5% Cifloxacin (Bayer Pharma, Puteaux, France) and 1% glutamine (Invitrogen). Heparin (2 IU/ml, Sanofi-Aventis, Paris, France) was added only to PLcomplemented medium to avoid clot formation. The culture medium was changed twice every week and the cultures were maintained in a humidified atmosphere with 5% CO2 at 37 C. When cells reached 80–90% confluence (passage 0, P0), they were detached using trypsin/EDTA and then replated at 1000 cells/cm2 (passage 1, P1). 2.3. Cell growth analysis To compare cell proliferation between the various culture conditions, at P1 MSCs were plated at 1000 cells/cm2 in a 25-cm2 flasks and, after 7 days of culture, trypan blue was used to count viable cells (in six replicates by two independent investigators). To estimate the duration of one mitosis (doubling time) we use the following formula: t/n, where t is the time for the MSC plated at 1000 cells/cm2 to reach 80% of confluence and n is the number of population doublings (means the number of mitosis to reach 80% of confluence). The number of population doubling is calculated by using the classical formula n ¼ log (y/x)/log 2 (where x is the number of cells originally plated and y, the number of cells at 80% of confluence) [28]. 2.4. Characterisation and differentiation of human MSCs The capacity of the PL- and the FBS-expanded MSCs to differentiate into the osteogenic and adipogenic lineages was determined. For this purpose, cells were seeded in 6-well plates. For osteogenic differentiation, at 25% confluence the media containing either PL or FBS were supplemented with 50 mM L-ascorbic acid-2phosphate and 10 mM b-glycerophosphate with or without 0.1 mM dexamethasone (Sigma, Saint Quentin Fallavier, France). On days 0, 7 and 14, cells were harvested, washed twice with HBSS (Invitrogen) and lysed for RNA extraction (Qiagen, see Quantitative Real-Time Reverse Transcription). On day 21, the monolayers were fixed in 70% ethanol for 1 h at 4 C and stained for 15 min with alizarin red-S (Sigma) at room temperature (RT). For adipogenic differentiation, at 80% confluence the media containing either PL or FBS were replaced by a high glucose medium (Invitrogen) supplemented with 10% FBS, 0.1 mM dexamethasone, 0.2 mM indomethacin, 0.01 mg/ml insulin and 0.5 mM IBMX. On day 21, the monolayers were fixed using 4% paraformaldehyde for 5 min at RT, and then stained for 15 min with 0.3% oil-red O (Sigma)/60% isopropanol. 2.5. Flow cytometry MSCs (P1) from six BM were resuspended in phosphate buffer containing 2% FBS with fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-coupled antibodies against CD105 (Caltag Laboratory, CA, USA), CD90, CD73, or CD45, or the corresponding mouse IgG1 isotype (all from Becton Dickinson and Company, Franklin Lakes, NJ, USA) for 15 min at RT. The cells were washed and examined using a FACScan flow cytometer. The data were analysed using the Cell Quest software (Becton, Dickinson and Company). Positive expression was defined as fluorescence greater than 95% of that of the corresponding isotype-matched control antibodies. 2.6. Quantitative real-time reverse transcription–polymerase chain reaction (RQ-PCR) Total mRNA was isolated from various MSC cultures on days 0, 7 and 14 after P1 using an RNeasy mini kit as described by the manufacturer (Qiagen). DNAse (Promega)-treated RNA was reverse transcribed with RT Superscript II (Invitrogen), and the cDNA was amplified with the TaqMan-Polymerase chain reaction (Applied Biosystems, Courtaboeuf, France) and monitored with the ABI Prism 7900 Sequence Detection System (PerkinElmer/Applied Biosystems, Rotkreuz, Switzerland). Amounts of cDNAs of interest (Table 1) were normalized to that of GAPDH (DCt ¼ CT DCT ). gene of interest CT GAPDH). Results are reported as relative gene expression (2 Table 1 Taq Man primers. GAPDH 0sterix (0sx, SP7) Runx2 ALP Osteopontin Osteocalcin (BGLAP) BSP Collagen type1 alpha1 Collagen type II alpha1 AP2 (FABP4) BMP2 c-Myc htert

Hs99999905 Hs00541729 Hs00231692 Hs00758162 Hs00960942 Hs00609452 Hs00173720 Hs00164004 Hs00156568 Hs00609791 Hs00154192 Hs00153408 Hs00162669

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2.7. Biomaterials and cell seeding Scaffolds of hydroxyapatite (65%) – beta tricalcium phosphate (35%) (HA/bTCP) presenting an average porosity of 65 5% (60 5% macroporosity and 40 5% microporosity) and a specific surface area of 0.8 m2/g were kindly provided by Ceraver (Roissy, France). Cubes (3 3 3 mm3) of these HA/bTCP scaffolds were maintained in culture medium supplemented with either PL or FBS at 37 C overnight. The HA/bTCP scaffolds were statically loaded with 3 105 MSC (in 50 ml culture medium) for 3 h. The MSC-HA/bTCP were then placed into untreated 24-well culture plates and cultured in either FBS or PL medium at 37 C in a 5% CO2 atmosphere for 7 days. Cell-free scaffolds were incubated under similar conditions and served as controls. 2.8. Animal model and implantation procedure Six (7 week-old) SCID mice purchased from Charles River laboratories (Chatillon, France) were used in this experiment in accordance with French law on animal experimentation. Isoflurane was used for anaesthesia. Subcutaneous dorsal pockets (0.5 cm incisions) were prepared on each mouse. In each pocket, one scaffold containing or not containing MSC cultured in either FBS or PL medium was implanted and the skin was closed by suture. After 7 weeks, the animals were euthanized by overdose of pentobarbital and the specimens excised and immediately put in buffered formol fixative. MSC-HA/bTCP cultured in either PL (n ¼ 8) or FBS (n ¼ 6) and HA/bTCP cultured in FBS (n ¼ 6) or PL (n ¼ 6) and subcutaneously implanted into nude mice were analysed by both non-decalcified and decalcified histology techniques. 2.9. Histology A histological procedure for non-decalcified bone was used for the following excised tissue specimens: seven MSC-HA/bTCP-PL, five MSC-HA/bTCP-FBS, five HA/bTCP-PL and five HA/bTCP-FBS. After fixation in 10% phosphate-buffered formalin, each specimen was rinsed in water, dehydrated in ethanol, cleared in xylene, and embedded in methyl methacrylate. Three sections were prepared from each specimen. Each section was ground down using the Exact Grinding System (Exact Aparatebau GmbH Norderstedt, Germany) and polished down to a thickness of 50 mm. These sections were then stained with either Stevenels’ blue or van Gieson picro-fuschin and visualized using standard light microscopy. Bone mineralization areas were counted on the three sections from each specimen. A histological procedure for decalcified bone was used for one excised tissue specimen of each group embedded in paraffin. Sections (5 mm) were stained with haematoxylin, eosin and saffranin (HES) and visualized using standard light microscopy. 2.10. Statistical analysis For cell growth analysis, all measurements were performed in triplicate by two independent investigators. Ten different bone marrows were used. Comparisons between the four experimental groups (a-MEM-LP or FBS and LP02-LP or FBS) were performed using the unpaired nonparametric Kruskal–Wallis test (GraphPad Software) with a p-value of 0.05 considered to be significant. Quantitative PCR was performed at least in duplicate for each of six different MSC samples analysed. Comparisons between the two experimental conditions (FBS and PL) were

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3. Results 3.1. Effect of PL on MSC growth The growth-stimulating activity of PL in primary MSC cultures was evaluated in two different media by measuring cell proliferation and comparing it to the standard medium aMEM 10% FBS cultures (aMEM-FBS). MSCs from six different bone marrows were isolated (P0) in four different media: aMEM-FBS and -PL, and LP02FBS and -PL, then plated at 1000 cells/cm2 (P1) and grown for 7 days. PL significantly increased MSC expansion in vitro in aMEM (2.8 fold) and in LP02 (3.5 fold) (Fig. 1a). In contrast in vitro expansion in LP02, a medium which contains anti-oxidant, was not significantly higher than that in aMEM (1.5 fold, p > 0.05; Fig. 1). To calculate the doubling time of the cell population (at P1), MSCs from 10 different donors were grown at a maximum of 80% confluence for 7–12 days. Our results showed a long MSCs doubling time in FBS media (52 14 h in aMEM-FBS and 42 7 LP02-FBS). In contrast, the PL media allowed a higher MSC proliferation as the doubling time is significantly reduced (32 4 h in LP02 and 34 7 h in aMEM, (p < 0.05)) (Fig. 1b). Thus, the presence of PL significantly shortened the doubling time in both media, confirming the value of PL for MSC amplification, independent of the composition of the medium. As the expansion rate was higher in LP02-PL than in aMEM-PL, MSC growth in LP02-PL (PL) was compared to that in the standard aMEM-FBS (FBS) medium for the rest of the study. 3.2. MSC characterisation The growth-promoting activity of PL influenced cell morphology, as assayed by FACS analysis and MSC counts when the cultures reached confluence. In six independent experiments, the cell density at confluence was 3-fold higher in the presence of PL, suggesting that PL-expanded cells were smaller (Fig. 2a). Forward scatter (FSC) FACS analyses of the six independent donors confirmed that cells were smaller in the presence of PL than FBS (mean FSC values were 426 112 for PL and 567 96 for FBS; Fig. 2b).

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Fig. 1. Cell proliferation in various media. (a) After 7 days of culture, cells were counted in six replicates by two independent investigators. Reported values have been normalized to aMEM 10% FBS. The fold induction of cell proliferation is shown. (b) Cell population doubling time (number of days of culture/number of population doublings). A Kruskal–Wallis test was performed. The significance of the difference from aMEM-FBS is represented: *p < 0.05; **p < 0.01; ***p < 0.001.


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Immunophenotypic characteristics of FBS- and PL-expanded MSCs were compared by flow cytometry. More than 98% of expanded MSCs in both media were strongly positive at P1 for CD90, CD105, and CD73, which are hallmarks of MSC (Fig. 2b). The cultures did not contain hematopoietic lineage cells, as indicated by the absence of CD45-expressing cells (Fig. 2b). The influence of PL on the osteogenic and adipogenic differentiation potential of MSCs was investigated after appropriate induction at the second passage (P2). Cells grown in PL or FBS deposited an extensive mineralized matrix when cultured for 3 weeks in an osteogenic medium, as demonstrated by strong alizarin red staining (Fig. 2c). These cells also efficiently differentiated into the adipogenic lineage, as indicated by Oil Red O staining of lipid droplets in the cytoplasm following culture in an adipogenic medium (Fig. 2d). Due to the potential risk of transformation associated with rapid cell growth, the expression of two cell transformation markers, c-myc and htert, was analysed. Relative to transformed human lineage cells (UT7 and 293 T) as positive controls, c-myc expression levels were very low, and htert was not expressed at all by MSCs at P1. Similar identical results were observed in both media (Fig. 2e). 3.3. Osteoblastic gene expression Osteoblastic gene expression was analysed after MSC amplification (P1) in PL and FBS media without adding a differentiation agent. MSCs from five or six different BM were analysed using quantitative RQ-PCR. The expression of ALP, BSP, Op, and BMP2 was significantly up-regulated in cells cultured in PL than in those of FBS (8, 8, 41, and 80 times higher expression, respectively, p < 0.01) (Fig. 3). No significant differences were observed for the

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In vitro mineralization, the last step of osteoblastic differentiation, is usually obtained after 21 days of confluent culture in the presence of osteogenic inducers such as AA, bGly and dexamethasone. Because PL is able to up-regulate late osteoblastic gene expression, we investigated whether 21 days of confluent culture in the presence of PL without adding osteoblastic agents was sufficient to induce in vitro mineralization. In the absence of osteogenic factors in the medium, no mineralization was observed with either FBS or PL (Fig. 4a). However, on day 14 the expression of Runx2 (mean of 1.8 0.6) and BSP (mean of 7.8 4.7) was higher when cells were maintained in PL medium (Fig. 4b). In the presence of minimal osteoblastic agents (AA and bGly), mineralization was obtained in both conditions (Fig. 4a). On day 14, we observed an up-regulation of Runx2 (4 1.6), ALP (31 28), BSP (24.6 17.9), and Oc (3.5 1.2) which correlated to the mineralization observed in the FBS condition. The expression of Op and BMP2 remained very weak, and was not significantly up-regulated (3 2.7 and 2.3 1.3, respectively). Expression of Osx was not observed on day 14 (Fig. 4b, and data not shown). In contrast to the mineralization observed in PL culture, the presence of AA plus bGly did not further induce Runx2 (1.8 0.8), ALP (1.6 0.4), Op (1.4 0.7), Oc (1.1 0.5) or BMP2 (2 1.4). On day 14, BSP was up-regulated (8.6 5.6) to levels comparable to those in untreated

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cells (7.8 4.7) (Fig. 4b). AA and bGly induced osteoblastic gene expression only when applied to MSCs grown in FBS, but the expression levels on day 14 were almost equivalent to the basal expression levels of these genes when MSCs were grown in PL. These results are consistent with PL by itself being sufficient to prime osteoblastic differentiation. 3.5. In vivo bone formation The osteogenic capacity of MSC cultured in either PL or FBS was investigated in vivo using ectopic implantation into immunodeficient SCID mice. Four groups were tested: (i) MSC-HA/bTCP cultured in PL medium (n ¼ 8, MSC-HA/bTCP-PL); (ii) MSC-HA/ bTCP cultured in FBS medium (n ¼ 6, MSC-HA/bTCP-FBS) and the HA/bTCP control groups incubated under similar media conditions but without cells (n ¼ 6). Seven weeks post implantation, the scaffolds were collected: one scaffold of each group was decalcified and the others were not decalcified to preserve mineralized bone formed within them. Minimal indication of ceramic resorption was observed after 7 weeks of implantation. There was no evidence of inflammatory reaction either in tissues inside the pores of the constructs or in peri-implant tissues (Fig. 5a, b and c). Bone mineralization was observed in all seven implanted MSCHA/bTCP cultured in PL, at a mean of five areas per scaffold

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Table 2 In-vivo bone mineralization. Scaffold incubated with

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(Table 2; Fig. 5a and d). Mineralized bone tissue was found both peripherally and in inner pores of the scaffolds (Fig. 5a). This new bone contained osteocyte-like cells and osteoblast-like lining cells at the surface and several blood vessels as observed in decalcified and non-decalcified sections (Fig. 5d and g). By contrast, only one of the five MSC-HA/bTCP cultured in FBS exhibited two areas of bone mineralization (Table 2). Three of the other five implants showed areas occupied by osteoid tissue (Fig. 5 e and h); the two others showed only fibrous and blood vessel tissues (data not shown). No bone formation but loosely organized connective tissues with numerous blood vessels were observed in all negative control HA/bTCP scaffolds incubated in either PL or FBS (Table 2; Fig. 5c, f and i). These results demonstrate that MSC expanded and cultured on HA/bTCP scaffolds in PL medium improved in vivo bone formation.

Fig. 5. Histological analysis of MSC-HA/bTCP scaffold in PL medium (a, d, g); -FBS medium (b, e, h) and control scaffold (c, f, i). Non-decalcified implants were embedded in polymethyl methacrylate and stained with Stevenels’ blue (nuclei) and van Gieson picro-fuschin (bone tissue) (a–f), arrow heads indicate mineralized bone area (a). Decalcified implants were embedded in paraffin and stained with haematoxylin (nuclei) and eosin–saffranin (cytoplasm and collagen) (g–i). Original magnification: 4 (a–c) and 10 (d–i). O ¼ osteoid; B ¼ organized bone; Ob ¼ osteoblast-like cells; Oc ¼ osteocyte-like cells; V ¼ blood vessels.

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4. Discussion Only small quantities of MSCs are present in total BM samples so in vitro cell amplification is necessary for cell therapy protocols. MSC culture conditions may influence bone formation, and osteogenic stimulatory agents are usually used to induce osteoblastic differentiation. This study analysed MSCs expanded in media in which PL replaced FBS in order to investigate their plasticity and ectopic bone formation capacity. We confirm that PL-expanded cells show a significantly higher proliferation rate [14] and demonstrate that PL induces MSCs into the osteogenic lineage. Moreover, we show that bone-forming capacity after in vivo ectopic implantation was greater for MSCs pre-induced in PL medium than in standard FBS medium. Recently, the use of PL as a culture supplement for MSC expansion has been suggested as a promising FBS substitute [14]. The expansion-promoting effect is likely due to the high concentration of natural growth factors that PL contains. Our data showed an improved proliferation of PL-expanded cells. The morphology of MSCs cultured in the presence of PL differed from that of MSCs grown in 10% FBS, as has been previously reported [29]; in particular, the cell size was smaller in PL cultures, possibly due to the higher proliferation rate. However, the higher proliferation rate and differences in cell morphology were not associated with abnormal c-myc and htert expressions, primary indicators used in safety assessments of MSC preparation. In addition, cells growing in FBS and PL stopped proliferating after 6–10 passages, depending on the sample (data not shown). Despite the differences in morphology and growth, MSCs cultured in PL and FBS media expressed similar phenotypic markers. Although PL has been extensively analysed, its role in osteogenic differentiation remains unresolved [11,20,30]. Preparation methods are an important determinant of the PL medium’s efficiency, as they can dramatically influence platelet and growth factor concentrations [31], and consequently the osteogenic capacity [32]. It should be noted that platelet growth factor levels vary considerably between donors and species, with the highest levels found in human PL, followed by goat PL and rat PL [12,33]. Consequently, results from different studies are difficult to compare. To provide a basis for future clinical applications, we investigated the effects of frozen human PL on human MSCs without adding exogenous components. We report that human PL improved osteogenic MSC differentiation. Unlike other studies, we used human PL-supplemented culture medium from the beginning of culture. MSC differentiation was studied at the molecular level without exogenous osteogenic inducers such as AA, bGly, dexamethasone, or BMP2 added to the culture medium. We found a significant up-regulation of several late osteoblastic genes, notably ALP, BSP, Op, and to a lesser extent, Oc, in the PL medium relative to control medium. Despite bone marrow sample heterogeneity, these genes (ALP, BSP, Op) were more strongly expressed (2-fold or more) in PL medium than FBS medium. No expression of either late adipogenic AP2 or chondrogenic Col II genes was detected in cultures in untreated PL medium at P1. When associated with aMEM medium, PL had a similar effect on the induction of late osteoblastic genes (data not shown). This supports PL having a role as a primer of MSC osteoblastic orientation, as has been suggested for human cell lines [23]. PL improves both cell growth and osteogenic differentiation. PL’s impact on MSC osteoblastic differentiation is supported by PL’s numerous growth factors (GF), which include BMP2-4-6, TGF-b1, IGF, bFGF, PDEGF, PF4, interleukin-1, and osteonectin (a major protein in mineralized bone), some of which are known to have osteo-inductive effects [12,16,34,35]. This cocktail of growth factors (GFs) affects multiple pathways, distinguishing PL from single recombinant GF, which act on single signal transduction pathways. The GF cocktail varies

between PL donors, but all our PL samples showed a similar capacity to induce osteoblastic gene expression. Our PL culture medium was supplemented with heparin to avoid clot formation. Heparin sulfate has mitogenic and osteogenic effects on MSCs via bone morphogenetic protein signalling pathways, resulting in a high Osx expression [36]. In our study, however, the heparin concentration used was not sufficient to induce Osx expression or mitogenic activation (data not shown); the mitogenic and osteogenic effects observed can be attributed to the PL. The transcription factors Runx2 and Osx are crucial early markers of commitment to the osteogenic lineage [37,38]. Runx2 up-regulates BSP [39] and Oc [40], the two major components of the bone extracellular matrix synthesized exclusively by osteoblastic cells. After amplification (P1), we observed that PL up-regulated late osteoblastic genes, but did not upregulate the early markers Runx2 and Osx. These findings suggest that PL induced late osteoblastic genes via a pathway independent of Runx2 and Osx. We cannot, however, exclude a transient expression of Runx2 and Osx at the beginning of the culture [41]. In addition to up-regulating late osteoblastic genes, PL medium also stimulated the expression of the strong osteo-inductive agent BMP2 [42,43]. Secretion of BMP2 by MSC could be an advantage for autocrine and paracrine induction for in vitro and in vivo osteoblastic differentiation. When cells were maintained at confluence for 2–3 additional weeks before mineralization analysis, we observed the following: i) In untreated PL medium, there was an up-regulation of Runx2 associated with an up-regulation of BSP, suggesting a new signal-like cell-to-cell interaction, including synergistic effects with PL when cells were confluent. The addition of osteogenic inducers, for example AA and bGly, had no influence on osteoblastic gene expression levels, probably due to the higher basal expression levels observed following P1. However, up-regulation of the osteoblastic gene was not associated with in vitro mineralization in PL medium. Mineralization was restored when a source of organic phosphate, b glycerophosphate, was added to the culture [44]. ii) In untreated FBS medium, no osteoblastic gene induction was detected on day 14 when cells were confluent. The up-regulation of osteoblastic gene expression was only detected after the addition of the exogenous inducers, AA and bGly, and was associated with mineralization at day 21, as was expected. We used an ectopic model of bone formation to investigate MSCPL-induced pre-osteoblastic differentiation in vivo. For MSC-HA/ bTCP cultured in PL and implanted into a vascularised environment in vivo, we observed bone formation in 100% of the implants. By contrast, only 50% of the MSC-HA/bTCP-FBS showed osteoid tissues and only one implant showed mineralization beginning (20%). No bone formation was observed with unloaded cell scaffolds. These findings in vivo are consistent with MSC having a role in neo-bone formation and confirm the importance of in vitro osteoblastic MSC pre-orientation for the initiation of bone formation in vivo [45]. Finally, our in vivo and in vitro results confirm the value of using PL as a pre-osteoblastic inducer, without the need for adding other osteogenic inducers such as the synthetic glucocorticoid dexamethasone. No differences in cell loading efficiency or proliferation were observed for HA/bTCP scaffolds seeded with MSC cultured in PL or FBS (data not shown), so it is unlikely that the difference in bone formation was due to different numbers of cells loaded onto the scaffolds. This further supports the idea that the major role of PL is to pre-induce MSC to the osteoblastic lineage. In our model system, scaffold loading with PL alone was not sufficient to induce bone formation. This observation differs from the findings of Kasten


et al., who studied bone formation in a cranial graft model [46]. The discrepancy is most likely because our ectopic model lacks osteoprogenitor cells able to graft onto the scaffold. 5. Conclusion Our in vivo data using an ectopic model and our in vitro quantitative PCR data regarding osteoblastic gene expression confirm that PL medium induces MSC cultures into osteoblastic differentiation pathways. PL can replace osteogenic agents in the preinduction of MSCs before in vivo implantation. Therefore, human PL provides a novel tool suitable for clinical-scale expansion of MSCs without using xenogenic substances such as FBS. Our findings provide evidence of the potential of MSCs cultured in PL medium to enhance in vivo bone regeneration and in particular for clinical treatment of pseudo-arthrosis.

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Acknowledgments [22]

This work was supported by EFS Ile de France, The French Research minister and Fondation de l’Avenir (grant ET6-432). The authors are grateful to E. Allaire, M. Gervais, JJ Lataillade and N Ortonne for helpful discussion and the animal platform facilities. The authors are grateful to the company Ceraver which kindly provided the ceramics.

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