The Journal of Bone and Joint Surgery 80:1745-57 (1998)
© 1998 The Journal of Bone and Joint Surgery, Inc.
The Chondrogenic Potential of Human Bone-Marrow-Derived Mesenchymal Progenitor Cells*
JUNG U. YOO, M.D. ,
TRACI S. BARTHEL, M.D.,
KEITA NISHIMURA, M.D.,
LUIS SOLCHAGA, PH.D.,
ARNOLD I. CAPLAN, PH.D.,
VICTOR M. GOLDBERG, M.D. and
BRIAN JOHNSTONE, PH.D., CLEVELAND, OHIO
Investigation performed at Case Western Reserve University, Cleveland
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Abstract
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Mesenchymal progenitor cells provide a source of cells for the repair of musculoskeletal tissue. However, in vitro models are needed to study the mechanisms of differentiation of progenitor cells. This study demonstrated the successful induction of in vitro chondrogenesis with human bone-marrow-derived osteochondral progenitor cells in a reliable and reproducible culture system.
Human bone marrow was removed and fractionated, and adherent cell cultures were established. The cells were then passaged into an aggregate culture system in a serum-free medium. Initially, the cell aggregates contained type-I collagen and neither type-II nor type-X collagen was detected. Type-II collagen was typically detected in the matrix by the fifth day, with the immunoreactivity localized in the region of metachromatic staining. By the fourteenth day, type-II and type-X collagen were detected throughout the cell aggregates, except for an outer region of flattened, perichondrial-like cells in a matrix rich in type-I collagen. Aggrecan and link protein were detected in extracts of the cell aggregates, providing evidence that large aggregating proteoglycans of the type found in cartilaginous tissues had been synthesized by the newly differentiating chondrocytic cells; the small proteoglycans, biglycan and decorin, were also detected in extracts. Immunohistochemical staining with antibodies specific for chondroitin 4-sulfate and keratan sulfate demonstrated a uniform distribution of proteoglycans throughout the extracellular matrix of the cell aggregates. When the bone-marrow-derived cell preparations were passaged in monolayer culture as many as twenty times, with cells allowed to grow to confluence at each passage, the chondrogenic potential of the cells was maintained after each passage.
CLINICAL RELEVANCE: Chondrogenesis of progenitor cells is the foundation for the in vivo repair of fractures and damaged articular cartilage. In vitro chondrogenesis of human bone-marrow-derived osteochondral progenitor cells should provide a useful model for studying this cellular differentiation. Furthermore, the maintenance of chondrogenic potential after greater than a billion-fold expansion provides evidence for the clinical utility of these cells in the repair of bone and cartilage.
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Introduction
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Mesenchymal progenitor cells have an important role in the repair of musculoskeletal tissues. Because bone marrow has been shown to contain pluripotential mesenchymal progenitor cells with the ability to differentiate into chondrocytes and osteoblasts when reimplanted in vivo4,17,21, much focus has been directed to the isolation and use of these cells. Long before the advent of the field of tissue engineering and modern repair concepts, it had been realized clinically that the mobilization and differentiation of these cells was important in the repair of damaged articular cartilage and fractured bones8.
The importance of the bone-marrow components in the repair of articular cartilage has been highlighted by the lack of repair of articular cartilage where the damage does not penetrate the subchondral bone8,9,22. The ineffectual repair is due to the inability of cartilage to heal itself and to the inaccessibility of the site to chondroprogenitor cells from the bone marrow. In contrast, if the damage to the articular cartilage extends beyond the subchondral bone, a repair process ensues in which mesenchymal progenitor cells migrate from the bone marrow into the injured site and undergo chondrogenic differentiation. However, analysis of both animal models and human biopsy samples has indicated that fibrocartilage, rather than true articular cartilage, is the predominant tissue synthesized8,9,30,31,40.
Both bone-marrow-derived and periosteal progenitor cells are involved in the repair of fractured bones37. Fracture repair that is accomplished by endochondral ossification and callus formation requires mobilization of the local cells that migrate into the fracture gap from the bone-marrow space. These cells differentiate into chondrocytes and then terminally differentiate into hypertrophic chondrocytes. If there is enough mechanical stability at the fracture site, vascular invasion occurs and the newly formed cartilage undergoes endochondral ossification. This process leads to the osseous bridging of the fractured bone.
To improve the repair of bone and cartilage, various biological implants have been developed that involve the use of progenitor cells. For example, tissues that contain osteochondral progenitor cells have been used as implants, whole bone marrow has been used in bone defects38,46, and perichondrium and periosteum have been used in cartilage defects14,25. Preparations of isolated and culture-expanded bone marrow and perichondrial cells containing progenitor cells have also been implanted in cartilage defects13,19,45, and bone-marrow cells have been implanted in bone defects24,34. In addition, many studies have shown that bioactive factors such as bone morphogenetic proteins induce the formation of bone at orthotopic and heterotopic sites in animal models27. Also, several studies have involved the use of bioactive factors in the repair of cartilage defects22,36,39. Although some of these implants have been reported to improve the repair of bone and cartilage, there is still much work to be done, especially in the field of cartilage repair, to produce a therapy that is clinically applicable.
The basis of these treatment strategies is the chondrogenic differentiation of mesenchymal progenitor cells, either within the implants or in response to the implanted bioactive factors. However, there is only limited knowledge about the differentiation potential of these cells and the process of chondrogenesis that they undergo. Successful in vitro chondrogenesis has been demonstrated with avian and embryonic mammalian cells and cell lines, and much valuable information has been gained from those studies2,3,11,15,20,47. However, until recently there was no in vitro system that induced the formation of cartilage from mesenchymal progenitor cells to allow direct study of the differentiation process. We recently described a culture sytem that facilitates the chondrogenic differentiation of rabbit bone-marrow-derived progenitor cells in vitro23. In the present study, we describe the application of that system to promote the chondrogenic differentiation of adult human bone-marrow-derived mesenchymal progenitor cells.
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Materials and Methods
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Methods for Studying the Chondrogenic Differentiation of Human Bone-Marrow-Derived Progenitor Cells
The procedures used for the removal, fractionation, and culture of human bone-marrow-derived mesenchymal progenitor cells (Fig. 1) will be described in detail in the following sections.
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Fig. 1 Diagram showing the removal, fractionation, and culture of human bone-marrow-derived mesenchymal progenitor cells. DMEM = Dulbecco modified Eagle medium, and FBS = fetal bovine serum.
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Removal of Cells and Formation of Colonies in Monolayer Culture
Human bone marrow was obtained from the iliac crests of fifty-five patients who were twenty-seven to seventy years old. A 14-gauge needle was used to penetrate the cortex of the bone, and five to eight milliliters of bone marrow was aspirated into a syringe containing 100 units of heparin. Dulbecco modified Eagle medium with 10 percent of a selected lot26 of fetal bovine serum was added to the aspirate, and a cell pellet was produced by centrifugation. The pellet was resuspended in five milliliters of Dulbecco modified Eagle medium with 10 percent fetal bovine serum, and it was fractionated on a Percoll gradient (70 percent initial density21). The upper fifteen milliliters of the gradient was collected, and the number of nucleated cells was determined. The cells were plated at 10 x 106 cells per 100-millimeter dish in low-glucose Dulbecco modified Eagle medium with 10 percent fetal bovine serum, and they were grown for eighteen to twenty-two days at 37 degrees Celsius, in 5 percent carbon dioxide, with the medium changed every four days. During primary culture, adherent cells formed colonies that were removed when they had expanded to cover approximately 90 percent of the plate. The cells were then trypsinized, counted, and either used for the cultures of cell aggregates (described later) or replated in monolayer culture.
Effect of Transforming Growth Factor (TGF)-ß1 on Cells Grown in Monolayer Culture
Passaged cells were seeded at 2 x 105 cells per thirty-five-millimeter dish. The cells were plated in chemically defined medium5 consisting of high-glucose Dulbecco modified Eagle medium with ITS+ Premix (Collaborative Biomedical Products, Bedford, Massachusetts), insulin (6.25 micrograms per milliliter), transferrin (6.25 micrograms per milliliter), selenious acid (6.25 micrograms per milliliter), and linoleic acid (5.35 micrograms per milliliter) with bovine serum albumin (1.25 micrograms per milliliter) and pyruvate (one millimole), ascorbate 2-phosphate (37.5 micrograms per milliliter), and 10-7-molar dexamethasone. Precoating of the dishes with fibronectin was necessary to ensure cell adhesion. The cultures were divided into six groups: control dishes without recombinant human TGF-ß1 (ten nanograms per milliliter [R and D Systems, Minneapolis, Minnesota]) or supplementation with 0.01, 0.05, 0.1, 0.5, or 1.0 nanogram of TGF-ß1 per milliliter. The culture dishes were incubated at 37 degrees Celsius in 5 percent carbon dioxide. The medium was changed every three days, and the cultures were analyzed on the twenty-eighth and thirty-fifth days. Duplicate dishes were rinsed twice with five milliliters of Tyrode salt solution (Sigma Chemical, St. Louis, Missouri), and they were fixed for fifteen minutes with a two-milliliter aliquot of 10 percent neutral buffered formalin. A two-milliliter aliquot of 1 percent alcian blue in 0.1-normal hydrochloric acid (pH 1) was added to each dish, and the dishes were incubated at room temperature overnight. The plates were rinsed twice with water and were examined for extracellular matrix that had stained positively for alcian blue. After visual examination of the stained plates, a one-milliliter aliquot of 10 percent cetyl pyridinium chloride was added to the plates and they were incubated for one hour on an orbital shaker at 37 degrees Celsius. After incubation, the absorbance at 601 nanometers was determined.
Suspension Culture of Cell Aggregates
Adherent cell colonies were trypsinized and counted, and the medium containing fetal bovine serum was then replaced with the chemically defined medium described earlier, with ten nanograms per milliliter of TGF-ß1 added. Aliquots of 2 x 105 cells in 0.5 milliliter of medium were then centrifuged at 500 times gravity in fifteen-milliliter polypropylene conical tubes. The pelleted cells were incubated at 37 degrees Celsius in 5 percent carbon dioxide.
Within twenty-four hours after incubation, the cells formed an aggregate that did not adhere to the walls of the tube. The medium was changed every two or three days, and cell aggregates were obtained at intervals of as long as twenty-one days. From twelve of the cell preparations, aliquots were also spun down and incubated in the chemically defined medium without TGF-ß1 or dexamethasone, or with either factor only.
Histological and Immunohistochemical Analysis
The cell aggregates either were frozen in embedding medium or were fixed in 10 percent formalin and embedded in paraffin. Some cell aggregates were fixed with 1 percent glutaraldehyde in phosphate-buffered saline solution, postfixed with 1 percent osmium tetroxide in phosphate-buffered saline solution, and embedded in Embed 812 epoxy resin (EMS, Washington, Pennsylvania). For histological evaluation, sections were stained with toluidine blue. For immunohistochemical analysis, the frozen sections were fixed for ten minutes in methanol after a brief immersion in distilled water to remove the embedding medium. For paraffin-embedded cell aggregates, the sections were first deparaffinized. Nonspecific antibody binding was blocked by incubating the slides in 5 percent bovine serum albumin in phosphate-buffered saline solution for one hour. The sections were then incubated with primary antibody, diluted in 0.5 percent bovine serum albumin in phosphate-buffered saline solution, for sixty minutes. The sections were probed with anti-collagen antibodies raised against type-II collagen (II-II6B3, developed by Thomas Linsemer and obtained from the Developmental Studies Hybridoma Bank, Department of Biological Sciences, University of Iowa, Iowa City, Iowa), type-X collagen (kindly provided by Gary Gibson, Henry Ford Hospital, Detroit, Michigan), and type-I collagen (Ab-1, Oncogene Research Products, Cambridge, Massachusetts). In addition, sections were immunostained with anti-glycosaminoglycan antibodies 5-D-4 (anti-keratan sulfate) and 2-B-6 (anti-chondroitin 4-sulfate) (both kindly provided by Bruce Caterson, University of Wales, Cardiff, United Kingdom). To facilitate antibody access and to create the 2-B-6 epitope, sections were predigested with chondroitinase ABC (Seikagaku America, Ijamsville, Maryland) at 0.1 unit per milliliter in 0.1-molar Tris acetate. Reactivity was detected with fluorescence microscopy after incubation for thirty minutes with a fluorescein-conjugated secondary antibody (anti-mouse Ig; Cappel, Aurora, Ohio) that had been diluted 1:400 in 0.5 percent bovine serum albumin in phosphate-buffered saline solution.
Extraction and Analysis of Collagen
Cell aggregates were radiolabeled with [3H]-proline (100 microcuries per milliliter) for twenty-four hours at selected times of culture. Ten cell aggregates were labeled each time and then were combined and mechanically broken up with a pellet pestle in microfuge tubes in a small amount of their labeling medium. The homogenate was then added to the combined remainder of their labeling medium, and an equal volume of 2x extraction buffer (two milligrams of pepsin per milliliter of 1-normal acetic acid, 1-molar sodium chloride) was added. After overnight incubation at 4 degrees Celsius with constant agitation, collagens were precipitated by adding sodium chloride to attain a final concentration of two moles and by incubating them overnight at 4 degrees Celsius. The precipitates were then pelleted by centrifugation at 40,000 times gravity, resuspended in 0.4-molar sodium chloride, 0.1-molar Tris, pH 7.4, 0.5 percent 3-[(3-cholamidopropyl)dimethylammonio]-1-propone-sulfonate (CHAPS), and free radiolabel was removed by repeated centrifugation in microconcentrators (5000-molecular-weight exclusion membrane) (Ultrafree; Millipore, Bedford, Massachusetts). The total incorporated radiolabel was assessed by liquid scintillation spectrometry. The newly synthesized, radiolabeled collagens were then analyzed by electrophoretic separation (7.5 percent isocratic gel with 4 percent stacking gel) and fluorography after fixation in 45 percent methanol, 10 percent acetic acid, and impregnation with 2,5-diphenyloxazole (22 percent in dimethyl sulfoxide). Collagen quantification was performed by cutting bands corresponding to the  -chains 1(II) and 1(I), which comigrate, and 2(I) and 1(X), respectively; dissolving them in 30 percent hydrogen peroxide; and subsequently performing liquid scintillation spectrometry. A radiolabeled collagen type-II and type-X -chain preparation (kindly provided by Gary Gibson) was used as a standard for the electrophoretic analyses.
Quantification of Proteoglycan Synthesis
Cell aggregates were radiolabeled for six hours with 35S-sulfate4 (100 microcuries per milliliter) at various time-points in low-sulfate (0.05-millimolar) Dulbecco modified Eagle medium with the chemically defined medium components. For each time-point, ten cell aggregates were labeled, collected into a single microfuge tube, and then mechanically broken up with a pellet pestle in extraction buffer (4-molar guanidine hydrochloride, 0.05-molar sodium acetate, pH 5.8, 0.5 percent CHAPS, with proteinase inhibitors) and incubated overnight at 4 degrees Celsius with constant agitation. Proteoglycans were then precipitated by adding absolute ethanol (precooled at -20 degrees Celsius) to a final concentration of 67 percent, and the samples were left at -20 degrees Celsius overnight. The precipitates were pelleted by centrifugation and were resuspended in 4-molar urea, 0.5 percent CHAPS, in phosphate-buffered saline solution, before removal of free radiolabel by repeated centrifugation with aliquots of fresh buffer in Centriplus 30 concentrators (Amicon, Beverly, Massachusetts). Non-specific binding was minimized by prespinning a solution of bovine serum albumin (100 micrograms per milliliter) through the membrane. The total amount of incorporated radiolabel was assessed by liquid scintillation spectrometry.
Assessment of DNA and the Number of Cells in Cell Aggregates with Time in Culture
The number of cells present in the cell aggregates at various times during the culture period was assessed with the CyQuant cell proliferation assay (Molecular Probes, Eugene, Oregon). Triplicate cell aggregates were obtained and were broken up separately with a pellet pestle in the working reagent made according to the manufacturer's instructions. The mixture was then transferred to a microtiter plate well, and the fluorescence was quantified on a Fluorimager (Molecular Dynamics, Sunnyvale, California). For DNA quantification, a standard curve was constructed with the DNA supplied with the kit. A second standard curve was constructed with counted aliquots of adherent bone-marrow-derived cells that were obtained at the end of primary passage but were not subjected to aggregate culture.
Characterization of the Proteoglycans Synthesized by Cell Aggregates
Cell aggregates that had been incubated for fourteen days were fragmented and treated as described earlier for the extraction of proteoglycans. The extracts were then buffer-exchanged into phosphate-buffered saline solution in Centricon 30 concentrators (Amicon), and the proteoglycan content was determined with a safranin-O spectrophotometric assay12. Aliquots of the cell aggregates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 4 to 20 percent gradient gels after chondroitinase ABC digestion (0.01 unit per milliliter, 37 degrees Celsius for one hour) and then were electroblotted onto Immobilon membranes (Millipore). Immunolocation of various proteoglycan components was then done with antibodies raised against the hyaluronan-binding region of aggrecan (polyclonal HABR, kindly provided by Michael Bayliss, Royal Veterinary College, London, United Kingdom), link protein (8-A-4, kindly provided by Bruce Caterson, University of Wales, Cardiff, United Kingdom), and decorin and biglycan (LF-30 and F-15, respectively, kindly provided by Larry Fisher, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland). The membranes were blocked with 5 percent bovine serum albumin in ten-millimolar Tris-buffered saline solution (pH 8.0, 0.15-molar sodium chloride, 0.05 percent Tween) before incubation with primary antibody diluted in Tris-buffered saline solution. Antibody binding was detected after incubation of the blots with alkaline phosphatase-linked anti-mouse Ig (Promega, Madison, Wisconsin) or anti-rabbit Ig (Sigma Chemical, St. Louis, Missouri) secondary antibodies diluted in Tris-buffered saline solution and subsequent addition of substrate (Nitro Blue Tetrazolium, 5-bromo-4-chloro-3-indoyl phosphate on 0.1-molar sodium chloride, five-millimolar magnesium chloride, 0.1-molar Tris, pH 9.55). Between each step, the blots were rinsed three times with 1 percent bovine serum albumin in Tris-buffered saline solution.
Effect of Passage on the Chondrogenic Potential of Bone-Marrow-Derived Cell Cultures
To examine the effect of continuous expansion of the adherent cell cultures on their differentiation potential, monolayer cultures were passaged twenty times, with replating at 0.5 x 106 cells per 100-millimeter dish each time. Aliquots of cells were taken at the first twelve passages and again at the twentieth passage, and they were used to create cultures of cell aggregates, which were obtained at seven, fourteen, and twenty-one days. The chondrogenic potential of the cell aggregates was evaluated by staining formalin-fixed, paraffin-embedded sections with toluidine blue.
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Results
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There was great variability in the concentration of nucleated cells in the bone-marrow samples, with concentrations ranging from 2 x 106 to 5 x 107 cells per milliliter. The age of the donor had no relationship with the number of cells per milliliter of bone marrow removed or with the ability of the culture-expanded cells to undergo chondrogenesis.
Effect of TGF-ß1 on Bone-Marrow-Derived Mesenchymal Cells Grown in Monolayer Culture
The cells that were cultured under control conditions (without TGF-ß1) or with low concentrations of TGF-ß1 (0.01 to 0.1 nanogram per milliliter) acquired a more polygonal shape (Fig. 2) and grew to form multilayered cultures. With higher concentrations of TGF-ß1 (0.5 or 1.0 nanogram per milliliter), the cells became more spindle-shaped and detached from the culture dishes. None of the monolayer cultures had any areas of alcian-blue-positive matrix at either time-point analyzed.
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Fig. 2 Monolayer cultures of bone-marrow-derived cells without recombinant human TGF-ß1 (control) (A) or with 0.1 (B) or 1.0 (C) nanogram of TGF-ß1 per milliliter.
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Changes in the Morphology of Bone-Marrow-Derived Mesenchymal Cell Aggregates with Time in Culture
The morphological changes in the cell aggregates were analyzed by examining epoxy resin-embedded sections stained with toluidine blue. On the first day, the aggregates consisted of cells that varied greatly in morphology, including their size and shape, but that tended to be oval to round (Fig. 3). Some of the cells were arranged in a syncytium, whereas others were individually delineated. At the periphery of the cell aggregates, a few of the cells were elongated (fusiform). On the fifth day, there was an increase in fusiform cells throughout the aggregates, but particularly at the periphery, where the cells tended to form a multilayered zone. Metachromasia increased, especially in the middle of the cell aggregates. By the seventh day, the center of the aggregates consisted of more plump, rounded cells. By the fourteenth day, the major difference was that the cells stood out as individual entities surrounded by metachromatic matrix.
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Fig. 3 Sections of epoxy resin-embedded cell aggregates cultured for one (A), five (B), seven (C), or fourteen (D) days (toluidine blue, x 128).
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By the seventh day, the cell aggregates that had been incubated without TGF-ß1 or dexamethasone, or with either factor alone, were much smaller than those that had been incubated with both factors. With more time in culture, most of the cell aggregates that had been incubated without TGF-ß1 (with or without dexamethasone) did not survive, such that the cell aggregates disappeared by the fourteenth day. Only the cell aggregates that had been incubated with TGF-ß1 alone survived until the fourteenth day. Toluidine-blue staining of sections of cell aggregates incubated with dexamethasone or TGF-ß1 alone revealed no evidence of matrix production or chondrogenic differentiation in any of them (Fig. 4).
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Fig. 4 Sections of cell aggregates cultured for fourteen days and treated with dexamethasone (A and B) or TGF-ß1 (C and D) alone (toluidine blue, x 64).
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In fifty-five preparations, cell aggregates were incubated in the presence of both TGF-ß1 and dexamethasone. Fifty of these preparations underwent chondrogenic differentiation, as shown by staining with toluidine blue. There were five failures: three were due to infection of the cultures (which were discarded without histological analysis), and it was not known why the cell aggregates of the other two preparations failed to survive until the fourteenth day. In the fifty successful preparations, the cell aggregates all had extensive metachromatic extracellular matrix formation.
Analysis of sections of the cell aggregates that had been incubated with both TGF-ß1 and dexamethasone and that underwent chondrogenesis showed that extracellular matrix was evident by the fifth day (Fig. 5, B). The extent of the matrix increased during the next eight to ten days. Concomitant with this sequence was an initial decrease in the size of the cell aggregates during the first five days of culture, with a gradual increase in size thereafter. Metachromasia of the matrix was obvious from the fifth day (Fig. 5, B) on. Typically, this metachromasia was first detected in a region of the cell aggregate that was three to six cell layers in from the periphery, but not initially in the center, and it usually formed a ring of cellular matrix within seven days (Fig. 5, C). By the fourteenth day, the metachromatic-staining matrix was seen throughout the cell aggregate except at the outermost layers, which contained flattened cells (Fig. 5, D).
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Fig. 5 Sections of paraffin-embedded cell aggregates cultured for one (A), five (B), seven (C), or fourteen (D) days (toluidine blue, x 32).
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Immunohistochemical Characterization of Changes in the Matrix of Cell Aggregates with Time in Culture
The sequence of chondrogenic differentiation that cells undergo includes a change in the types of collagen that are synthesized and secreted. Initially, the highly cellular aggregates contained type-I collagen (Fig. 6, section 1-B), as would be predicted from the fibroblastic morphology of the cells in primary monolayer culture. Neither type-II nor type-X collagen was detected on the first day. However, type-II collagen was typically detected in the matrix by the fifth day and sometimes it was seen as early as the third day. The region of type-II immunoreactivity corresponded to the metachromatic-staining region described earlier. Immunodetection of type-X collagen occurred very early, but it appeared cell-associated until the seventh day, when it was also detected in the matrix. By the fourteenth day, type-II and type-X collagen were detected throughout the cell aggregates, except for the outer region of flattened cells (Fig. 6, sections 4-A and 4-C). Type-I collagen was still detected in the inner part of the cell aggregate, but the immunoreactivity was of much greater intensity in the outer, flattened cell region (Fig. 6, section 4-B).
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Fig. 6 Fluorescein-conjugated immunohistochemical analysis of frozen sections of cell aggregates cultured for one (1), five (2), seven (3), or fourteen (4) days. The sections were immunostained with antibodies reactive to epitopes within type-II (A), type-I (B), and type-X (C) collagen (x 32).
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Quantification of Changes in the Cells and the Matrix During Chondrogenesis of Cell Aggregates
The DNA content of the cell aggregates was quantified at various times during culture. It was established that the DNA content decreases over the first three days but is unchanged during the following eighteen days (Fig. 7, A). When a standard curve of pre-aggregate cells was used in the assay, the results were essentially the same because both curves were linear. The incorporation of 35S-sulfate4 into newly synthesized aggregate matrix molecules (which is essentially a measurement of proteoglycan synthesis) increased with time (Fig. 7, B). This would be predicted from the changing morphology of the cell aggregates—that is, the development of a substantial metachromatic matrix.
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Fig. 7 Changes, with time in culture, in DNA content (A) and in incorporation of 35S-sulfate4 per cell (B) of the aggregates. cpm = counts per minute.
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Analysis of the Major Types of Collagen in the Extracellular Matrix of Cell Aggregates
Immunohistochemical analysis of sections of the cell aggregates indicated that there was type-I, type-II, and type-X collagen in the aggregates and that the profiles of each type altered with time in culture (Fig. 6). To quantify these changes, pepsin-digested, salt-precipitated collagen extracts of the cell aggregates on the tenth and seventeenth days were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the newly synthesized, radiolabeled collagen profiles were analyzed. Fluorography of the gels indicated that type-I collagen was still being synthesized on the seventeenth day. Type-X collagen was not visible in the electrophoretically separated extract of the cell aggregates from the tenth day, but it was easily detected by the seventeenth day. Collagen 1(II) chains migrate with 1(I) chains, so the assessment of type-II collagen synthesis was done by quantification of the radioactive bands separated by electrophoresis. The bands were cut from the silver-stained gel and were solubilized for liquid scintillation spectrometry. The ratio of 1(I) to 2(I) chains is 2:1, so the 1(II) content was assessed by calculating the counts remaining after subtraction of the 1(I) content from the total counts within the upper band. The ratio of newly synthesized type-II, type-I, and type-X collagen was 3:2:1 on the seventeenth day.
Detection of Proteoglycans in the Cartilaginous Matrix Synthesized by Cell Aggregates
Immunohistochemical studies of the proteoglycans in extracts of cell aggregates were performed with use of core protein-specific antibodies, after the electroblotting of chondroitinase-digested, sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated extracts onto Immobilon membranes. Both the hyaluronan-binding region of aggrecan and link protein were detected in extracts of the cell aggregates (Fig. 8), providing evidence that large aggregating proteoglycans of the type found in cartilaginous tissues had been synthesized by the differentiating cells. The small proteoglycans, biglycan and decorin, were also detected in the extracts. Immunohistochemical staining with antibodies specific for chondroitin 4-sulfate and keratan sulfate (Fig. 9) indicated the distribution of proteoglycans throughout the extracellular matrix of the cell aggregates. The epitopes were localized predominantly in the metachromatic-staining region of the cell aggregates at all of the analyzed time-points.
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Fig. 8 Immunolocation of epitopes within the hyaluronan-binding region of aggrecan (A), decorin (B), biglycan (C), and link protein (D) in extracts of cell aggregates from the fourteenth day separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a blotting membrane.
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Fig. 9 Fluorescein-conjugated immunohistochemical analysis of frozen sections of cell aggregates cultured for one (A and B), seven (C and D), or fourteen (E and F) days with anti-glycosaminoglycan antibodies 5-D-4 (A, C, and E) or 2-B-6 (B, D, and F) (x 32).
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Chondrogenic Potential of Multiple-Passaged Bone-Marrow-Derived Mesenchymal Cells
Bone-marrow-derived cell preparations were passaged in monolayer cultures as many as twenty times, with cells permitted to grow to confluence after each passage. The chondrogenic potential of the cells was maintained throughout the experiment, as judged by the morphological appearance of the sections of cell aggregates created by cells taken at each passage. All cell aggregates had a very similar appearance: an extensive, metachromatic-staining matrix by the fourteenth day, with hypertrophic cells present (Figs. 3 and 5).
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Discussion
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Clinically, the most important sites of chondrogenesis are articular cartilage and healing fractures of bones. It is well accepted that the repair tissue arises from the differentiation of neighboring (local) mesenchymal progenitor cells. Both the periosteum and the bone marrow contain such cells, which have the ability to differentiate into both chondrocytes and osteoblasts4,17,18,29,32. The repair of fractured bone requires mobilization of the local bone-marrow cells, which are carried to the fracture gap as a result of bleeding from the bone marrow6,28. If the local and systematic milieu is adequate, these cells accumulate, multiply, and differentiate into chondrocytes and then terminally differentiate into hypertrophic chondrocytes. If there is appropriate mechanical stability at the fracture site, the newly formed cartilage undergoes endochondral ossification and osseous bridging of the fractured bone ensues eventually6,28. The repair of articular cartilage defects extending beyond the subchondral bone also requires mobilization and differentiation of mesenchymal progenitor cells. If the damage to the articular cartilage extends beyond the subchondral bone, the pluripotential mesenchymal cells migrate into the site of injury. Some of these cells differentiate to chondrocytes, although fibrocartilage rather than hyaline cartilage is formed8,9,30,31,40.
There is particular interest in mesenchymal progenitor cells of bone in tissue repair because of the extensive reserve of these cells, their ease of removal, and their expandability in culture7. However, despite the interest in regeneration of articular cartilage and repair of fractures, the mechanism of chondrogenesis is poorly understood. The complexity of in vivo conditions hinders identification of the factors that are important in the transformation of mesenchymal progenitor cells into chondrocytes and those that are important in the control of their terminal differentiation into hypertrophic chondrocytes. The development of an in vitro culture system that promotes the chondrogenic differentiation of these cells presents an opportunity to study the complexities of chondrogenesis in cells that are of potential clinical utility. Avian and embryonic mammalian cells and cell lines that undergo chondrogenic differentiation have been used to explore the mechanism of chondrogenesis2,3,11,15,20,47. However, until recently, attempts to induce the chondrogenesis of postnatal mammalian mesenchymal progenitor cells were not successful. In a recent study, we described a culture system that promotes the chondrogenic differentiation of postnatal rabbit bone-marrow-derived progenitor cells23. In the present study, we describe the use of that system to induce the chondrogenic differentiation of mesenchymal progenitor cells from adult human bone marrow.
Successful in vitro chondrogenesis required chemically defined serum-free culture conditions. No chondrocytic markers were detected in the cell aggregates at the initiation of culture in the chemically defined medium, indicating that these cells were not chondrocytes. Within three to five days, the presence of a metachromatic-staining matrix, the chondrocytic appearance of cells, and the immunochemical detection of type-II collagen clearly demonstrated that the tissue generated by these bone-marrow-derived cells was cartilage. The immunodetection of epitopes for large and small proteoglycan core proteins, link protein, and glycosaminoglycans indicated the presence of cartilage proteoglycans.
Attempts to initiate chondrogenesis monolayer cultures under the same conditions failed, but differentiation was induced when the cells were cultured as aggregates. In this respect, the cell aggregates resemble the precartilage condensation of cells in the forming limb bud16. The requirement for this high cell density suggests that cell interactions are very important in the system that we developed. The interactions between cells as well as the lack of interaction between cells and substratum were noted by Solursh to be important for in vitro chondrogenic differentiation and maintenance of chondrocyte phenotype41.
It is of interest that the outermost part of the cell aggregates contained several layers of flattened cells that did not undergo chondrogenic differentiation. They formed a perichondrium-like structure around the cell aggregate and produced type-I collagen, but no type-II or type-X collagen was detected. Because the initial cell preparations that were used for forming the cell aggregates were heterogenous, it is possible that these cells were a separate fibroblast-type phenotype and that a cell-sorting phenomenon occurred during formation of cell aggregates. Perichondrium-like elongated cells have also been found at the periphery of embryonic chick limb-bud-cell aggregates, formed in suspension cultures42,43 and in high-cell-density embryonic chick limb-bud-cell cultures35. These cells may have some role in the chondrogenesis of the cell aggregate, possibly producing paracrine factors, but their importance is unknown at this time.
The addition of TGF-ß1 was necessary to induce the differentiation of the bone-marrow-derived progenitor cells to chondrocytes. The requirement for TGF-ß1 in this system is not surprising. In embryonic cartilage, TGF-ß1 is found in abundant amounts and may play an important role in the chondrogenic transformation of primitive mesenchymal condensations10. In the postnatal organism, fracture repair also requires local factors such as members of the TGF-ß1 superfamily of growth factors. Such factors may be locally derived from the degradation of bone matrix, or they may be brought to the injured site by cells involved in inflammation33. In the culture system used in the present study, the addition of TGF-ß1 provided the appropriate external signal for chondrogenic differentiation to occur.
The chondrocytic cells in this culture system differentiated into their terminal phenotype, the hypertrophic chondrocyte, as indicated by changes in the cell morphology and by detection of type-X collagen. Alkaline phosphatase activity is also greatly increased during aggregate chondrogenesis23. The terminal differentiation of these cells into hypertrophic chondrocytes also occurs in vivo in the reestablishment of subchondral bone in the repair of articular cartilage or in the osseous union of fractured bone. In the culture system used in the present study, there was a very short time-period between the appearance of type-II collagen in the matrix and the appearance of type-X collagen. This was observed previously in the chondrogenesis of chick embryonic cells in culture1. The in vitro findings are consistent with the results of immunohistochemical analysis of fracture repair, in which type-X collagen also appeared quickly after the detection of the type-II collagen in the matrix44.
Studies involving the implantation of progenitor cells into cartilage defects13,19,25,45 have indicated that these cells may have utility in that type of repair. However, hyaline-like cartilage was not consistently generated in the studies done to date. Part of the rationale for these cellular implants is that the in vivo environment should provide all of the signals that are required for the appropriate differentiation of the implanted-cells. However, it is possible that the signals that are present during development and growth of tissues such as articular cartilage are no longer present in adults. For the successful production of articular cartilage, it may be necessary to provide these cells with signals, such as TGF-ß1 or related bioactive factors, to promote differentiation.
We also found that the chondrogenic potential of human bone-marrow-derived mesenchymal progenitor cells is retained through multiple passages. This is an important attribute if these cells are to be used for the repair of musculoskeletal tissue because in vitro expansion of the cells by multiple passaging may be necessary to provide sufficient cells for a successful implant. Studies of the chondrogenic ability of multiple-passaged mammalian bone-marrow-derived cells have not been reported, to our knowledge. Multiple passaging of rat bone-marrow-derived cells does decrease their osteogenic potential18, but the osteogenic potential of human bone-marrow-derived cells appears to be retained with extensive expansion in culture7.
In the present study, we used a culture system that promotes the chondrogenic differentiation of human bone-marrow-derived mesenchymal progenitor cells. This system should prove useful by helping investigators to determine the molecular mechanisms involved in chondrogenesis. Furthermore, the system may allow the identification of new markers that are useful in defining the normal process of cartilage formation and regeneration.
NOTE: The authors thank Yee Hsee Hsieh and Amad Awadallah for excellent technical assistance, Dr. James E. Dennis for preparing the histological sections embedded in epoxy resin, and the spine surgeons of the Department of Orthopaedics at the University Hospitals in Cleveland for supplying preparations of bone marrow. We also thank Gary Gibson, Ph. D., David Carrino, Ph. D., and John Carter, M. D., for all of their advice and assistance.
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Footnotes
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*No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article. Funds were received in total or partial support of the research or clinical study presented in this article. The funding sources were National Institutes of Health Grants AR-44390 (B. J.) and AR-37726 (V.M.G.).
Departments of Orthopaedics (J. U. Y., T.S.B., K. N., V. M. G., and B. J.) and Biology (L. S. and A. I. C.), University Hospitals, Case Western Reserve University, 11100 Euclid Avenue, Cleveland, Ohio 44106. E-mail address for Dr. Johnstone; bxj9@po.cwru.edu.
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Abstract
Introduction