Short-Term BMP-2 Expression Is Sufficient for In Vivo Osteochondral Differentiat - 《stemcells》2004-2005文献干细胞之家



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a Inserm U475, Montpellier, France;; p0 I9 G; D3 |$ a5 ^8 w

b Molecular Pathology Laboratory, Hebrew University-Hadassah Faculty of Dental Medicine, Jerusalem, Israel;& ]. T: f2 k# t' T5 y
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c Institut Gustave Roussy, Villejuif, France;6 A- e0 k, [. {; S3 w; U0 j
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d H?pital Lapeyronie, Service d’Immuno-Rhumatologie, Montpellier, France7 \/ P4 \* p& ?4 \8 |; b/ a
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Key Words. Chondrocytes ? Osteocytes ? BMP-2 ? Tet-Off system ? Vascular endothelial growth factor ? Intra-articular ? Stromal progenitor cells7 z" @- l' Y+ \1 T0 L

Dani豕le No?l, Ph.D., Inserm U475, 99 Rue Puech Villa, 34197 Montpellier Cedex 5, France. Telephone: 33-04-67-63-62-74; Fax: 33-04-67-04-18-63; e-mail: noel@montp.inserm.fr

ABSTRACT# ~( V, Y& z6 R

Articular cartilage is frequently damaged in different pathological situations such as osteoarthritis, rheumatoid arthritis, and trauma. In chondrodysplasia, a rare genetic disorder leading to dwarfism, defects in the growth plate cartilage result in reduced bone growth. Due to its poor capacity of self-regeneration, new approaches based on cellular therapies have been investigated. Human autologous chondrocyte transplantation has proved to be successful for the repair of deep cartilage defects in the knees of patients subsequent to trauma . Although treatment resulted in the formation of new cartilage that was similar to normal, it seemed to be limited to defects resulting from trauma and could not be extended to other osteochondral abnormalities such as osteoarthritis, probably owing to the pathological state of implanted chondrocytes.
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More recent attempts of cartilage regeneration have been made using mesenchymal stem cells (MSCs) originating from the bone marrow . These progenitor/stem cells are pluripotent and can differentiate into multiple lineages such as myoblasts, adipocytes, osteocytes, and chondrocytes . Such MSCs have thus been successfully used to repair large full-thickness defects created in the femoral condyle of rabbit . However, although the subchondral bone was completely regenerated without loss of the overlying articular cartilage, progressive thinning and incomplete integration of the repaired tissue within the host cartilage were observed at 24 weeks, suggesting the need for local growth/differentiation factors.

Bone morphogenetic proteins (BMP) belong to the transforming growth factor ? (TGF-?) superfamily, which participates in the regulation of cell migration, adhesion, multiplication, and differentiation throughout the life span of the organism . Some of these growth factors display osteoinductive properties that induce ectopic bone and cartilage formation in vivo. Among these growth factors, BMP-2, BMP-4, and BMP-7 (also called osteogenic protein-1) have been largely used as recombinant proteins for their ability to repair bone defects in different animal models . Based on this property, MSCs have been engineered to locally deliver BMP-2 in order to induce their differentiation into bone cells. Hence, using ex vivo adenoviral gene transfer, BMP-2-expressing MSCs were shown to form bone when injected into muscle . More importantly, BMP-2-producing progenitor cells can be used successfully to heal segmental femoral defects in syngeneic Lewis rats  and radial segmental defects in mice . Fibroblast-mediated growth factor gene therapy has also been used for generating cartilage repair after transplantation into knee joints of mice . In that study, it was suggested that secretion of BMP-2 by ectopic fibroblasts stimulated chondroid tissue and osteophyte formation, probably acting through a local stimulus on cells present in the joint. A similar approach was used in rabbits using TGF-?1-expressing fibroblasts . Interestingly, the results indicated that the local production of TGF-?1 induced proliferation of chondrocyte and/or chondrocyte precursors inside the joint, leading to a gradual replacement of ectopic fibroblasts by rabbit chondrocytes, and generation of a fully differentiated hyaline cartilage at 6 weeks.
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BMP-2 is also proposed to exhibit an angiogenic effect both in vitro, using a chick chorioallantoic membrane assay, and in vivo, after subcutaneous implantation of BMP-2-expressing MSCs that induced formation of blood vessels in mice . In adults, angiogenesis is essential for pathological processes such as tumor growth and also for physiological processes such as tissue repair and regeneration during wound healing. Among the known angiogenic growth factors, the main effectors are the vascular endothelial growth factor (VEGF), the angiopoietin family, and their receptors . In juveniles, VEGF is required for endochondral bone formation and longitudinal growth . It has been shown that BMPs stimulate angiogenesis through the production of VEGF by osteoblasts , both acting synergistically to increase endochondral bone formation .

Taking into account the importance of the environmental factors on the differentiation capacities of stem cells in vivo, we investigated whether BMP-2-engineered MSCs form bone in the particular synovial/articular cartilaginous environment, as it has been reported in other ectopic sites. To this end, we used the C9 cell clone derived from the C3H10T1/2 murine mesenchymal progenitor cell line , expressing the human (h)BMP-2 gene under control of the Tet-Off system in an attempt to timely regulate the expression of the differentiation factor. These cells were injected into the intra-articular space of mouse knee joints, and we showed that BMP-2-expressing MSCs formed hypertrophic cartilage that was rapidly replaced by bone by a process resembling endochondral ossification. These results suggest that the endogenous factors secreted into the joint space had no detectable effect on the differentiation capacities of adult MSCs. Importantly, we showed that a short-term expression of the growth factor (BMP-2 in our experiments) is both necessary and sufficient to irreversibly induce bone formation.1 q. ]& [' t/ ~! S' e

MATERIALS AND METHODS

Engineered C9 MSCs Undergo Chondrogenesis In Vitro
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As the engineered C9 cells expressing the hBMP-2 gene under control of the Tet-Off system have been previously shown to differentiate in vitro into osteoblasts , we wanted to determine whether they also kept their chondrogenic potential. To this aim, C9 cells were cultured in micropellets either in presence or in absence of Dox to, respectively, repress or induce BMP-2 secretion. In these culture conditions, absence of a differentiation factor, such as BMP-2, will not induce chondrogenesis. The expression of chondrocyte-specific markers (aggrecan, type IIB and type X collagens) were checked by RT-PCR. All three markers were detected in C9 cells cultured in absence of Dox, whereas type IIA collagen mRNA was repressed (Fig. 1). During differentiation, the type II procollagen mRNA switches from type IIA in undifferentiated MSCs to type IIB splice variant in chondrocytes . In contrast, in C9 cells cultured with Dox and in control C3H10T1/2 cells, genes related to chondrogenic differentiation markers were undetectable (aggrecan) or slightly expressed (type X collagen) and the type IIA collagen splice form was predominant. Thus, C9 MSCs have undergone differentiation toward chondrocytes and secreted the components of the cartilaginous matrix under chondroinductive culture conditions.
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Figure 1. In vitro differentiation of C9 cells toward chondrogenesis. RNA was extracted from C3H10T1/2 cells and C9 cells, with or without BMP-2 (± Dox), cultured in chondroinductive conditions for 21 days. Expression of the chondrocyte-specific markers: aggrecan (Agg), type X collagen (Col X), and type IIA and IIB collagens (Col IIA and Col IIB) were analyzed by semiquantitative RT-PCR using the GAPDH gene as control.6 b' W1 }; p8 m# E+ \' d
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BMP-2-Expressing MSCs Generate Cartilage and Bone after Intra-Articular Injection6 Y3 A+ E! P. ^
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To test whether MSCs could undergo spontaneous differentiation in the intra-articular space, we injected na?ve C3H10T1/2 MSCs into CB17-SCIDbg mice knee joints. We showed that most of the cells have settled onto the synovial membrane or cartilage, leading to a mesenchymal-like tissue but never cartilage or bone (Fig. 2A). The injection of immortalized mesenchymal cells (NIH-3T3) led to tumor growth that invaded the joint and formed a protuberance at the knee surface but neither cartilage nor bone tissue (data not shown). We then tested the behavior of increasing numbers of C9 MSCs (104, 105, or 106 cells) in the articular environment of mice maintained without Dox diet to induce BMP-2 expression. On day 21 after cell injection, histological analysis showed that whatever the number of cells, both cartilage and bone could be detected in injected knees. The size of the newly formed tissue was related to the number of injected cells. The new chondroid/bony tissue was preferentially localized in the recesses of the synovial membrane or in the suprapatellar region. The neocartilage showed characteristic chondrocytic cells surrounded by large lacunae and revealed a high content in proteoglycans as shown by strong safranin O staining (Fig. 2C). Intense remodeling of the matrix that began to calcify was also evidenced, and mineralization was confirmed by x-ray (data not shown). The bone trabecula had developed and bone marrow had filled the medullary cavity.; }1 Y7 K: t. ^1 p% t6 D- \+ S. o

Figure 2. Intra-articular differentiation of C9 cells. Cartilage and bone generation was monitored 21 days after injection of C9 cells into the knee joints of CB17-SCIDbg mice. Hematoxylin-eosin-safranine O staining of the joint sections was performed after inclusion in paraffin. A) Control mice injected with C3H10T1/2 showed mesenchymal-like tissue formation inside the intra-articular space (arrows); B) By repression of BMP-2 expression, C9 cells did not differentiate (arrows); C) In presence of BMP-2, C9 cells formed hypertrophic cartilage, characterized by high content in proteoglycans (orange staining of the matrix) and cells included in large lacunae (arrow), and bone that was filled with bone marrow cells (arrowheads); D) A section of the neotissue showing differentiated C9 cells; E) Same section as in (D) observed in fluorescence showing C9 cells that have been previously labeled with the CM-DiI fluorescent dye. All the observations were performed at magnification x100.; Q2 Q/ z9 v' N8 v$ ?, y
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A similar process was observed when C9 cells were injected in the tibialis anterior muscles of mice (data not shown). To confirm that the neotissue was originated from the injected cells, we labeled the cells before implantation using CM-DiI fluorescent dye. The fluorescent cells were found in the areas where the cartilage/bone neotissues were observed (Fig. 2D, 2E). As control, mesenchymal-like tissue was observed in mice receiving C9 cells combined with Dox diet (Fig. 2B), suggesting a tight regulation of the Tet-Off system. In control contralateral knees, formation of new cartilage or bone structures was never observed (data not shown).- S( w  I! ?3 V# E. q" I5 K2 M) J2 s
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We then monitored the differentiation process at earlier time points (Fig. 3). As soon as day 10, calcification could be easily detected by x-ray in mice injected with 106 cells and by histology with 105 or 104 injected MSCs (data not shown). Moreover, the neotissue formation increased over time as observed by x-ray at day 60 (Fig. 3).  T9 M: I6 t9 N8 W
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Figure 3. Kinetics of bone formation following C9 cell injection. Presence of calcified tissue was visualized by x-ray analysis as electron-dense areas in the knee joints of mice. A) On day 10, slightly mineralized tissue was observed in front of the knee joint (arrow). B) On day 21, more intensively calcified tissue was visualized in the suprapatellar localization (arrow). C) On day 60, intense ossification could be detected inside the entire joint limited by the synovial membrane (arrows).' v. F1 S! {) h, _1 a* D' V: r; b
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Intra-Articular Differentiation of BMP-2-Engineered MSCs Mimics the Process of Endochondral Ossification8 C$ Q6 R# i: L$ {9 G+ t
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Since bone formation was obvious 10 days following C9 cell injection, we monitored by histology the differentiation process from day 2–10. On day 2 and day 4 post-injection, MSCs were detected either lining the synovial membrane or forming mesenchymal condensations in the intra-articular spaces (Fig. 4A, 4B). On day 6, some mesenchymal condensations were still present and cartilaginous tissue resembling hypertrophic cartilage was observed as characterized by large round cells included in large lacunae (Fig. 4C). On day 8, large areas of hypertrophic-like cartilage were seen in the intra-articular space (Fig. 4D). On day 10, bony tissue was detected, with bone marrow invasion in some cases (data not shown). Immunohistological analysis revealed the expression of type II and type X collagens in the developing tissue as early as day 6 after C9 implantation. The type II collagen was poorly secreted, while type X collagen, specific for hypertrophic cartilage, was highly expressed in the chondroid tissue at day 8 (Fig. 4E, 4F). This differentiation program resembles the process of endochondral ossification in which a cartilaginous matrix, constituted of hypertrophic chondrocytes, is progressively replaced by a mineralized matrix.

Figure 4. Monitoring of the differentiation process of C9 cells at early time points. Following C9 cell injection, mice were euthanized on day 2, 4, 6, or 8. Knee joints were processed for histology and stained by hematoxylin-eosin. A) On day 2 after injection, C9 cells were localized in the intra-articular space forming mesenchymal-like cell condensations; B) At day 4, prechondrogenic mesenchymal condensations could be observed; C) On day 6, prehypertrophic cartilage characterized by cells in a lacuna was seen but mesenchymal-like tissue was still present; D) On day 8, large areas of typical hypertrophic cartilage could be observed. Such areas of hypertrophic cartilage were stained with anti-type X collagen- (E) or anti-type II collagen- (F) specific antibodies by immunohistochemistry.0 `% e( p& S+ g% k# p" v) l! v
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Short-Term Expression of BMP-2 Irreversibly Induces-Bone Formation

To determine whether BMP-2 expression is required during the overall osteochondral differentiation process, we took advantage of the tetracycline-regulated transgene expression by C9 cells. Mice were injected with CM-DiI-labeled C9 MSCs, and the secretion of BMP-2 was induced during day 2, 4, 6, or 8, and then repressed by addition of Dox in the drinking water until the end of the experiment. On day 21, histological sections of injected knees were performed. Secretion of BMP-2 for the first 4 days was not sufficient to induce MSC differentiation. Few C9 cells were detected as individual cells lining the synovial membrane or forming a mesenchymal-like tissue (Fig. 5A). On the contrary, BMP-2 expression for 6 days induced formation of hypertrophic cartilage and bone filled with bone marrow (Fig. 5B). Similar results were obtained when BMP-2 was expressed for 8 days. In most cases, the labeled C9 cells were mainly localized in the hypertrophic cartilaginous tissue and, more rarely, in the bony tissue without any preferential localization. Some areas of the new bone were completely devoid of fluorescent cells (data not shown). Altogether, these observations showed that, in this model, a 5–6 day period of BMP-2 expression was sufficient to trigger MSC differentiation and to lead to bone formation./ @) E) v- u: v5 E

Figure 5. Short-term expression of BMP-2 is sufficient to induce bone formation in the knee joints. Mice were injected with C9 cells and BMP-2 expression was repressed after different time intervals by addition of Dox in the drinking water until day 21. Histological analysis was performed at the end of the experiment by hematoxylin-eosin staining. A) Expression of BMP-2 for 4 days was not sufficient to induce MSC differentiation as a mesenchymal-like tissue was present in the joint; B) A 6-day period of BMP-2 expression was necessary to form bony tissue filled with bone marrow in the intra-articular space." J/ I  O0 ^+ x( j( u. z& b
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Decreased Secretion of VEGF During In Vitro MSC Differentiation
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During the process of endochondral bone formation, secretion of VEGF by hypertrophic chondrocytes is an important step involved in the replacement of cartilage by bone . We thus investigated whether secretion of BMP-2 by C9 cells could regulate VEGF production during their in vitro chondrogenic and osteogenic differentiation. We first determined the levels of VEGF produced in cell supernatants under proliferative culture conditions. Expression of VEGF was 148 ± 56 pg by 105 C3H10T1/2 cells in 24 hours versus 106 ± 20 pg and 99 ± 16 pg by C9 cell monolayers, with and without BMP-2 expression, respectively. Indeed, in nondifferentiating conditions, MSCs expressing BMP-2 secrete similar levels of VEGF to the na?ve cells (statistically not significantly different using the nonparametric Mann-Whitney test)., ~- H' h$ h. p' @5 W
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We then compared VEGF secretion when MSCs were induced toward osteogenic or chondrogenic differentiation by BMP-2 expression. In both cases, VEGF production dramatically decreased shortly after induction of the differentiation. During osteogenesis, we observed a dramatic decrease to undetectable levels by day 10, whereas during chondrogenesis, a weak but sustained production of VEGF in the range of 15 pg/pellet/24 hours was detected during the overall differentiation period (Fig. 6). Because dexamethasone is known to repress VEGF secretion , we tested the behavior of MSCs in the absence of dexamethasone in terms of differentiation potential and VEGF production. In osteogenic conditions, VEGF levels were maintained around 40 pg/105 cells/24 hours, but MSCs rapidly stopped proliferating and stayed scattered (data not shown). In the case of chondrogenesis, VEGF secretion was comparable to that obtained in the presence of dexamethasone and stayed stable over time (data not shown). In both conditions, differentiation occurred (data not shown). Independently of the presence of dexamethasone, induction of osteogenesis and chondrogenesis highly decreased VEGF secretion by differentiating MSCs.
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Figure 6. In vitro secretion of VEGF by C9 cells induced to chondrogenic or osteogenic differentiation. The levels of VEGF in culture medium were determined by specific ELISA. Chondrogenic differentiation was induced by culture of MSCs in micropellet (each point corresponds to six mixed supernatants). Results are representative of three separate experiments.0 y$ q4 A, f) J2 \6 P9 G7 q
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Neoangiogenesis Blockade Is not Sufficient to Inhibit Bone Formation
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Since it has been shown that BMP-2 exhibits an angiogenic effect that could participate in in vivo bone formation, we investigated the role of neoangiogenesis in our model of MSC differentiation. We tested whether inhibition of vascularization could delay or inhibit bone formation, resulting in increased cartilage formation. To this aim, we transduced C9 cells in vitro with an adenoviral vector expressing the antiangiogenic K3HSA fusion protein before intra-articular cell injection. First, the efficiency of transduction was estimated by measuring the K3HSA molecule produced in culture supernatants of infected C9 cells (Fig. 7A). The K3HSA fusion molecule was secreted in supernatants of transduced C9 MSCs at the level of 47 ± 12 ng/106 cells/24 hours but not in supernatants of nontransduced or control vector-transduced C9 cells (Fig. 7A). We then implanted the transduced C9 cells in the knee joints of mice and measured the quantity of K3HSA present in the serum (Fig. 7B). Stable expression of the exogenous protein could be determined throughout the experiment in the serum of injected mice (up to 700 ng/ml). No secretion of K3HSA could be detected in mice that received either nontransduced C9 cells or cells transduced with a void adenoviral vector (data not shown). Both radiological and histological analysis confirmed that transduced C9 cells had undergone differentiation, and ossification of the knee joints was observed. Immunohistochemical analysis of joint sections with an anti-smooth actin antibody did not reveal variation of blood vessel number in the new cartilaginous and bony tissues or in the surrounding tissues (data not shown). These results showed that in our conditions, an antiangiogenic agent, such as the K3HSA molecule, had no detectable influence on bone generation from engineered MSCs.  ~" y2 M: V6 \3 f2 i1 M. b) O2 d
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Figure 7. Detection of the K3HSA protein. A) In culture supernatants of C9 cells infected with the recombinant Ad-K3HSA adenoviral vector. The level of the K3HSA protein was determined by K3-specific ELISA 1 day after infection with the recombinant adenovirus (Ad-K3HSA) or a void vector (Ad-CO1). Nontransduced C9 cells were used as control (NT). Results are expressed on a pool of supernatants from eight culture dishes. B) In the serum of mice injected with Ad-K3HSA-transduced C9 cells. C9 cells were injected into the joints of CB17-SCIDbg mice (n = 10) and BMP-2 expression was induced by omitting Dox in the drinking water. Results are expressed in ng/ml as the mean ± standard deviation.
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DISCUSSION

We are grateful to Denis Greuet for excellent animal care, Mich豕le Radal (Centre de Recherches et de Lutte contre le Cancer Val d’Aurelle, Montpellier, France) for histological work, and the breast radiography group, headed by Professor Taourel, from Lapeyronie Hospital in Montpellier, who performed the radiographies. This work was supported in part by a grant from the Association Fran?aise contre les Myopathie (A.F.M.) and the research program Ing谷nierie Tissulaire from the INSERM and CNRS French Research Institutional Organizations.
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