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Multipotential Mesenchymal Stem Cells from Femoral Bone Marrow Near the Site of Osteonecrosis

^a Department of Internal Medicine,
^b Department of Orthopedics, and
^c Department of Dentistry, National Taiwan University Hospital and National Taiwan University, College of Medicine, Taipei, Taiwan;
^d Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan

Key Words. Mesenchymal stem cells • Bone marrow • CFU-F • Multilineage differentiation • ß1-integrin • Femoral head osteonecrosis

Correspondence: Ching-Chuan Jiang, M.D., Ph.D., Department of Orthopedics, National Taiwan University Hospital. No. 7, Chung-Shan South Road, Taipei, Taiwan (100). Telephone: 886-2-23123456 ext 5273; Fax: 886-2-23939632; e-mail: ccj{at}ccms.ntu.edu.tw

	ABSTRACT

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Stem cell-based therapies for degenerative disorders and injuries are promising in the new era. Multipotential mesenchymal stem cells (MSCs) from bone marrow (BM) are on the leading edge because they are easy to expand in culture while maintaining their multilineage potential. In vitro assessment of the chondrogenic and osteogenic potentials of cultured MSCs has been established, and the BM used in those experiments was exclusively from healthy donors via iliac crest aspiration. It is unknown whether human marrow obtained from femurs also contains these multipotential MSCs. We collected marrow from proximal femurs of two patients undergoing total hip replacement surgery for femoral head osteonecrosis and isolated and culture expanded MSCs to about 20 population doublings. These cells were homogeneously positive for ß1-integrin. When pelleted into aggregates and cultured in a medium containing transforming growth factor-ß3 for 14 days, the cells began to express mRNA for aggrecan and collagen type II and to deposit immunoreactive collagen type II and sulfated proteoglycans in the matrix, hallmarks of chondrogenic differentiation. These MSCs could also be differentiated into osteocytic lineage in vitro, as shown by increased expression of alkaline phosphatase activity and deposition of mineral content onto culture plates. These results indicate that femoral BM obtained during hip surgeries also contained multipotential MSCs. These data imply that direct replacement therapy using MSCs from in situ marrow may be possible in the future and that an MSC bank may be established by using marrow from this approach, bypassing the necessity for iliac marrow aspiration from healthy donors.

	INTRODUCTION

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Because of the lack of vascularity and paucity of cellularity, articular cartilage damaged by disease or trauma has a limited capacity for regeneration [1–3]. Using techniques of tissue engineering, artificial cartilage fabricated in vitro has been applied for the repair and regeneration of damaged cartilage [4]. A number of studies have shown that bone marrow (BM) stroma-derived mesenchymal stem cells (MSCs) represent a promising candidate cell type for cartilage and bone tissue engineering instead of primary chondrocytes or osteocytes, which are difficult to culture and expand. MSCs are multipotential cells, able to differentiate into tissues of mesenchymal lineages, including bone, cartilage, fat, tendon, muscle, and stroma [5].

Marrow stromal cell cultures are often defined as plating of BM and cultivation of nonhematopoietic adherent cells on culturing vessels. A standard in vitro assay for BM stromal cell activity is the fibroblastic colony-forming unit (CFU-F) assay in which adherent fibroblastic cells are cultured by plating BM cells either directly or following gradient separation [6]. It has been proposed that these marrow stromal cells are the progeny of MSCs [7]. Standard procedures to enrich MSCs from BM rely on Percoll gradient centrifugation [8–10]. In this study, we directly plated BM nucleated cells, which were the remaining cells following lysis of red blood cells, and culture expanded the adherent cells. We have previously reported that cells isolated from human BM in this way have the capability to give rise to progeny of osteocytes, indicating their multipotentiality [11]. In this study, we determined both the chondrogenic and osteogenic potential of these marrow stromal cells.

Most studies on chondrogenic and osteogenic differentiation of human MSCs have been done using BM aspirated via the iliac crest in healthy donors [3, 12–14]. However, it is unknown whether the marrow located near the sites of osteochondral defects also contains these multipotent cells. If this were true, there would be a therapeutic advantage in the future to using MSCs in the operating room that were obtained from BM at a site near the defect directly for replacement therapy of osteochondral defects. To assess the chondrogenic and osteogenic potential of marrow stromal cells isolated from BM procured via the proximal femurs during total hip replacement surgery in patients with femoral head osteonecrosis, we examined the differentiation characteristics of these culture-expanded stromal cells. Our results show that stromal cells in the femoral marrow from patients with osteonecrosis maintained their chondrogenic and osteogenic potential, even in a 72-year-old patient.

	MATERIALS AND METHODS

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Bone Marrow Procurement and Culture Expansion of Stromal Cells
After written informed consent was obtained from patients, BM samples (5-10 ml) were procured from proximal femurs during total hip replacement surgery. Two patients were included in this study. Their clinical characteristics are described in Table 1. The marrow was mixed (1:9 v/v) with a buffer containing 0.8% ammonium chloride and 10 µM EDTA (Sigma; St. Louis, MO; http://www.sigmaaldrich.com) for 10 minutes to lyse red blood cells. The remaining nucleated cell suspension was centrifuged at 500 g for 10 minutes, and the pellets were resuspended in Dulbecco’s modified Eagle’s medium-low glucose ([DMEM-LG] GIBCO; Grand Island, NY; http://www.lifetech.com) supplemented with 10% fetal bovine serum ([FBS] HyClone; Logan, UT; http://www.hyclone.com), 10 U/ml penicillin G, and 10 µg/ml streptomycin (GIBCO). The lot of the FBS used here was the same as that which we used to culture and differentiate MSCs into osteocytic lineage [11]. The cell suspensions in culture medium were cultured in 25-cm² flasks at 37°C in a humidified atmosphere containing 5% CO₂. Culture medium was changed every 3 to 4 days. Fibroblastic colonies formed on the plates were further expanded to 150-cm² flasks. When cells grew to 80% confluence, they were harvested and designated as passage 1. These cells were further expanded with 1:3 splitting. Cells at passage 3 (after about 20 population doublings) were used in this study.

Micropellet Formation and Chondrogenic Induction
Cells in culture were trypsinized and counted. Aliquots of 2 x 10⁵ cells in 0.5 ml of chondrogenic medium were pelleted at 500 g in 15-ml polypropylene conical tubes. Chondrogenic medium consisted of DMEM-LG containing ITS⁺ Premix (BD Biosciences; Bedford, MA; http://www.bdbiosciences.com), 1 mM of sodium pyruvate, 0.1 mM of L-ascorbic acid-2-phosphate, 10^-7 M of dexamethasone (Sigma), and 10 ng/ml of recombinant human transforming growth factor-ß3 (TGF-ß3) (R&D Systems; Minneapolis, MN; http://www.rndsystems.com). The ITS⁺ Premix contains 6.25 µg/ml of human recombinant insulin and transferrin, 6.25 ng/ml of selenous acid, 1.25 mg/ml of bovine serum albumin, and 5.35 µg/ml of linoleic acid. The cell pellets in conical tubes were incubated in a humidified atmosphere at 37°C in 5% CO₂.

One day later, the cells in pellets became aggregated and detached from the wall of tubes. Half of these day 1 pellets were taken from the tubes for experiments described below. The other pellets were maintained in culture in the same medium, with a medium change every 2 to 3 days. On day 14, the remaining pellets were harvested. The pellets were either embedded in OCT compound (Sakura Finetechnical Co.; Tokyo, Japan) and stored at -80°C or directly stored at -80°C until used for RNA extraction.

Histochemical Staining of Pellet Sections
The cryostats were sectioned at a 7-µm thickness and fixed in acetone for 10 minutes. For immunohistochemical staining, endogenous peroxidase activity of the sections was blocked with 0.3% H₂O₂ in methanol for 10 minutes. Sections were rinsed with water and then with Tris-buffered saline (TBS) solution containing 0.05 M Tris and 0.15 M NaCl at a pH of 7.6. To enhance protein antigen accessibility, the sections were incubated in 0.1 M Tris/acetate, pH 7.6, containing 40 mU/ml of chondroitinase and 1% bovine serum albumin (Sigma) at 37°C for 30 minutes. Blocking serum (10% rabbit serum) was used to block the nonspecific binding background. The sections were then incubated with anti-type-II-collagen goat polyclonal antibodies (Santa Cruz Biotechnology; Santa Cruz, CA; http://www.scbt.com) at a 1:50 dilution for 60 minutes at 37°C, and rinsed with TBS. They were then incubated with horseradish peroxidase-conjugated rabbit anti-goat secondary antibodies (Chemicon; Temecula, CA; http://www.chemicon.com) for 30 minutes. The immunoreactive areas were developed chromogenically with 3-amino-9-ethylcarbazole (DAKO) and counterstained with Mayer’s hematoxylin (Sigma).

For alcian blue staining, the slides were stained with alcian blue (Sigma) solution, pH 2.5, for 5 minutes and then washed in water.

Two-Dimensional (2D) Osteogenic Differentiation
To determine whether the marrow stromal cells also had osteogenic potential, we tried to differentiate these cells in plates to osteocytic lineage. The cells were seeded into 24-well plates at 3 x 10⁴ cells/well in the original medium (DMEM containing 10% FBS). The cells began to be incubated in osteogenic medium 24 hours after plating, and then changed with the same medium every 3-4 days. Control cells were maintained in the original medium. The osteogenic medium consisted of original medium plus 100 nM dexamethasone, 10 mM ß-glycerophosphate, and 50 µM L-ascorbic acid-2-phosphate (Sigma). After 14 days, the cells in 24-well plates were fixed with methanol at -20°C for 5 minutes and subjected to alkaline phosphatase and von Kossa staining.

For alkaline phosphatase staining, the fixed cells were washed with dH₂O for 1 minute and stained with a mixture of Fast Violet B salt and Naphthol AS-MX (3-hydroxy-2-naphtoic acid 2, 4-dimethylanilide) phosphate alkaline solution (Sigma), according to the manufacturer’s instructions, for 30 minutes, counterstained with Mayer’s hematoxylin for 10 minutes, and rinsed in tap water.

For von Kossa staining, the fixed cells in wells were incubated with 2% silver nitrate (Sigma) under direct light from a 60-W lamp for 1 hour. They were then fixed with 2.5% sodium thiosulfate (Sigma) for 5 minutes, counterstained with 0.33% neutral red (Sigma) for 5 minutes, and rinsed in tap water.

RNA Extraction and RT-PCR
Total RNA was extracted from cultured cells or the micropellets in 1.5-ml microcentrifuge tubes using Rezol reagent (Protech Technology; Taipei, Taiwan; http://www.bio-protech.com.tw) with homogenization. The lysates were extracted with chloroform, and total RNA was precipitated with isopropanol. One microgram of total RNA was reverse transcribed using random hexamers and reverse transcriptase ([RT] Promega; Madison, WI; http://www.promega.com). The first-strand cDNA product was subjected to polymerase chain reaction (PCR) using oligonucleotide primer pairs for type I and type II collagen, aggrecan, and a housekeeping gene, ß-actin (Table 2). The PCR protocol was an initial denaturation at 95°C for 3 minutes followed by 30 cycles of 94°C for 40 seconds, 55°C for 30 seconds, and 72°C for 45 seconds. PCR products were analyzed by electrophoresis in 2% agarose gels containing ethidium bromide and visualized with a charged-coupled device camera.

	RESULTS

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View larger version (147K): [in this window] [in a new window]   Figure 1. Photomicrographs of marrow stromal cells in primary culture. A) Cells in a CFU-F with scattered spindle shape were growing on the flask 7 days after plating of BM cells (scale bar = 100 µm). B) The cells were further cultured for another 2 weeks when they became confluent (scale bar = 40 µm).

View larger version (41K): [in this window] [in a new window]   Figure 2. Flow cytometry analyses of passage 3 cells from both patients. The filled areas represent the distribution of cells stained by the respective antibodies; the open areas are control cells without staining. Percentages in parentheses indicate the percentages of cells positively stained by the respective antibodies in the flow cytometry analyses.

View larger version (112K): [in this window] [in a new window]   Figure 3. Shown are type II collagen immunohistochemical staining of sections of micropellets that were cultured in chondrogenic medium for 1 and 14 days. A, B, and C are pellets from patient 1, and D, E, and F are from patient 2. A and D are from day 1, and B, C, E, and F are from day 14. In B and E, heavily stained areas are indicated by open arrows. C and F are magnified photos of the boxed areas in B and E, respectively. Arrows in C and F indicate spotty and lightly stained areas. Scale bars in A, B, D, and E = 100 µm and in C and F = 40 µm.

View larger version (111K): [in this window] [in a new window]   Figure 4. Alcian blue staining of the micropellets that were cultured in chondrogenic medium for 1 (A and C) and 14 (B and D) days. Pellets of A and B were cultured from BM cells of patient 1, while pellets of C and D were from patient 2. In the day 14 pellets, alcian blue staining became positive compared with day 1 pellets. Scale bar = 40 µm.

View larger version (53K): [in this window] [in a new window]   Figure 5. RT-PCR analysis of the expression of mRNA for type I and type II collagen and aggrecan in the 2D cultures and in pellets after chondrogenic induction. In 2D cultures and in both day 1 and day 14 pellets, expression of type I collagen was obvious and not different. While the expression of type II collagen was undetectable in 2D cultures and in day 1 pellets, it increased dramatically in day 14 pellets. The expression level of aggrecan was low in 2D cultures, but greater in day 1 pellets, and this level was maintained at 14 days. Loading of PCR products of ß-actin served as an internal control. Control = cells cultured two-dimensionally in DMEM containing 10% FBS.

View larger version (165K): [in this window] [in a new window]   Figure 6. Representative photomicrographs of alkaline phosphatase and von Kossa staining of cells from patient 2.Cells were incubated either without (A and C) or with (B and D) dexamethasone, ß-glycerophosphate, and L-ascorbic acid 2-phosphate for 14 days. A and B show staining for alkaline phosphatase activity. C and D show von Kossa staining for the deposition of calcium phosphate on the vessel. Alkaline phosphate activity was stained red, as shown in B; deposited calcium phosphate was stained black, as shown in D. Scale bars = 100 µm.

	DISCUSSION

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For regeneration therapy of tissues, which are damaged either by trauma or degeneration, stem cells with lineage differentiation potential are obviously superior to the mature cells of those tissues. The main reason is that stem cells have a greater capability than mature cells to proliferate in vitro. Moreover, these ex vivo-expanded stem cells could be readily turned into the desired cell types under the influence of cues from the in vivo microenvironment or from in vitro signals. This is especially true for MSCs as they could be easily obtained from BM and maintained in their undifferentiated phenotype for as long as 30-40 population doublings without losing their multilineage potential [16, 17]. After they were injected systemically, they could be incorporated into bone, cartilage, lung, spleen, brain, skin, and marrow 2.5 months later [18], following the differentiation guidance by the respective tissue environment. In vitro assays of differentiation have also been clearly established for bone, cartilage, and adipose tissues by addition of respective differentiation signals [14].

To isolate multilineage potential stromal cells from BM for culturing, a separation method using Percoll gradient has been proposed [8–10]. Recently, the gradient method to isolate multipotential MSCs from BM was challenged by showing, based on histology and RT-PCR, no qualitative differences in differentiative ability to osteogenic, chondrogenic, and adipogenic lineages between marrow stromal cells derived from standard Percoll gradient or from direct plating of supernatants of marrows after centrifugations at 600 g for 6 minutes [19]. In that study, the authors did not compare the phenotypic characteristics between cells isolated from both methods. In the present study, we isolated the stromal cells from the pellets of marrow after centrifugation at 500 g for 10 minutes. We did not culture the cells in the supernatants, thus we might have lost some smaller multipotential stromal cells with slower voyage in centrifugation. Nevertheless, the stromal cells in the centrifugation pellets were clearly shown here to have multipotentiality. It is possible that MSCs in the BM were heterogeneous in size with different stages of commitment. It would be interesting to correlate cell size, phenotyping, and lineage potentiality of the stromal cells. This knowledge would help establish a differentiation pedigree of stromal cell system from the beginning of the ultimate mesenchymal stem cells.

In contrast to hematopoietic stem cells and their progeny, the lineage progression of the surface antigens in MSCs has not been well defined. Several antibodies have been used to recognize MSCs. For example, Stro-1 antibody has been used to enrich mesenchymal precursors in the BM [20] and as a marker of those cells [21]. Stro-1⁺ cells isolated by magnetic immunobeads have been shown to be capable of differentiating into multiple mesenchymal lineages, including hematopoiesis-supportive stromal cells with a vascular smooth muscle-like phenotype, adipocytes, osteoblasts, and chondrocytes [22]. However, ex vivo-expanded marrow stromal cells lacking Stro-1 have also been shown to possess a full differentiation capability into osteocytic, chondrocytic, and adipocytic lineages [23]. Monoclonal antibodies of SH2 and SH3 were developed to show a specific reactivity to CFU-F in human BM [24]. SH2 antibody turned out to be reactive to an epitope on endoglin (CD105) [25]. In the present study, cells from both patients were all CD105^–. Thus, to date, there are still no monospecific and unique antibodies to unequivocally identify these progenitor cells [26]. Flow cytometry analyses in the present study revealed that 100% of the cells from both patients were positive for ß1-integrin. The other markers in order of positive rate were transferrin receptor, VCAM, and c-kit. As expected, these cells were negative for hematopoietic markers of CD34 and CD45. It has been reported that LFA-3, NCAM, 3-integrin, and Thy-1 were positive in mesenchymal stromal cells [16], but they were negative in our cells. The inconsistency of the reported surface markers on mesenchymal stromal cells may be caused by diverse population origin, different stage of commitment, the ex vivo culturing conditions, or just the different site of origin of BM, such as from femurs in this study.

Among the positive surface antigens, ß1-integrin might have a significant role in the chondrogenesis of these stromal cells. Integrins are heterodimeric molecules on cell surfaces, consisting of noncovalently associated and ß subunits. They play an important role in mediating cell-cell and cell-matrix interactions. These interactions are crucial for cell migration, differentiation, and adhesion during embryogenesis and development [27–31]. The in vitro chondrogenesis assay of marrow stromal cells depended on the micropellet aggregation method, which was performed in this present study. The aggregation would induce a close and unique pattern of cell-cell and cell-matrix interactions [32]. ß1-integrin has been demonstrated to be concentrated in adhesion plaques of the chondrocytes plated on type I collagen, type II collagen, and fibronectin substrates [33]. The adhesion of chondrocytes on these substrates can be completely blocked by ß1-integrin antibody [34]. Moreover, in vitro chondrogenesis using mesenchymal cells from limb buds of day-12 mouse embryos has been shown to be completely inhibited by ß1-integrin, but not by 1- or 5-integrin antibodies [35]. Taken together, our data suggested that ß1-integrin might play a significant role in the in vitro chondrogenesis of BM stromal cells.

As hematopoietic stem cells (HSCs), MSCs in BM also maintain a level of self-renewal and give rise to mesenchymal lineage cells [7]. The self-renewal capacity of these cells argues that these cells may not age and may provide progeny throughout the lifespan of an organism. But recent experimental results have shown evidence of aging in HSCs [36, 37] and in marrow stromal progenitor cells [38, 39]. Yet other reports demonstrated that osteogenic progenitor cells were maintained during aging [40, 41]. Although CFU-F efficiency was not determined in the marrow of our two patients, the present study showed, by in vitro chondrogenic and osteogenic assays, that marrow stromal cells isolated from patients as old as 72 years (patient 2) still maintained their multilineage potential.

In conclusion, results in the present study indicated that the direct use of MSCs from BM at a site of skeletal-cartilage defect might be possible for a novel replacement therapy in the future. This approach may improve the techniques to reconstruct the skeletal-cartilage defects with a better outcome. Another implication of this study is that banking of MSCs from BM may be established using marrows from patients without hematological disorders, who are undergoing orthopedic surgeries, at least during total hip replacement surgery. During the total hip replacement surgery, marrow is drained from proximal femurs, and is routinely discarded as waste. Taking advantage of using these marrows to isolate MSCs to establish a cell bank may bypass the necessity for volunteer marrow donors.

	ACKNOWLEDGMENT

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We thank the Gene Research Lab (Taipei, Taiwan) for technical assistance in flow cytometry analyses. This work was supported by a grant from National Taiwan University Hospital (NTUH-90A13).

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