Bone and cartilage formation in diffusion chambers by subcultured cells derived from the periosteum

Bone, 11, 181-188 (1990)

8756-3282/90 $3 .00 + .00 Copyright © 1990 Pergamon Press pie

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Bone and Cartilage Formation in Diffusion Chambers by Subcultured Cells Derived from the Periosteum H . NAKAHARA,' S . P BRUDER, 2 S . E . HAYNESWORTH,2 J . J . HOLECEK, 2 M . A . BABER,2 V. M . GOLDBERG3 and A . I . CAPLAN 2 Skeletal Research Center, Department of Biology, and Department of Orthopaedics, Case Western Reserve University, Cleveland, Ohio USA ' Visiting Scientist from Department of Orthopaedic Surgery, Osaka University Medical School, Osaka, Japan . 2 Skeletal Research Center, Department of Biology, Case Western Reserve University . s Department of Orthopaedics, Case Western Reserve University . Address for correspondence and reprints: University, Cleveland, OH 44106, USA .

Arnold 1. Caplan, Skeletal Research Center, Department of Biology, Case Western Reserve

cursor cells in bone marrow stroma . They also reported that these cell populations can be passaged many times in culture without loss of their osteogenic potential, as exhibited when again tested in implanted diffusion chambers (Friedenstein et al . 1987) . These observations have been confirmed and extended by others who, using diffusion chambers, have reported the osteo-chondrogenic potential of bone marrow cells from a variety of animal sources (Rosin et al . 1963 ; Friedenstein et al . 1970 ; Ashton et al . 1980 ; Bruder et al . 1988 ; Johnson et al . 1988) and also human (Bab et al . 1988) . The periosteum has also been reported to contain mesenchymal progenitor or stem cells capable of differentiating into either osteoblasts or chondrocytes as evidenced by studies of embryonic bone development (Fell 1925 ; Gardner 1971 ; Pechak et al . 1986a, 1986b), fracture healing (Ham 1930), and free periosteal tissue transplantation (Oilier 1859 ; Burman and Umansky 1930) . However, dispersed periosteal cells have not been tested in implanted diffusion chambers, although free periosteal flaps have been tested in this manner. Rosin et al . (1963) reported that 33 .5% of the test diffusion chambers were positive for bone and/or cartilage when loaded with free rat periosteal flaps . More recently, Jaroma and Ritsila (1988) reported the correlation of diffusion chamber pore size with differentiation and proliferation of periosteal cells of free rabbit periosteal flaps with higher positive incidence of bone and/or cartilage formation at larger pore sizes . We report here the development of methods, involving enzymatic liberation of periosteal cells from young chicks, their introduction into cell culture, and their mitotic expansion and subsequent subculturing . When once-subcultured periosteal cells are inoculated into a subcutaneous site in athymic mice, these cells and their descendants differentiate into osteoblasts or chondrocytes, eventually giving rise to bone tissue in vivo (Nakahara et al . in press) . With these data as a base, the osteo-chondrogenic potential of freshly isolated and then subcultured periosteal cells was assessed in diffusion chambers implanted into the peritoneal cavity of athymic mice . The objective of this study was to test whether these cultured cells give rise to bone

Abstract Periosteal cells were enzymatically isolated from the tibiae of young chicks, introduced into cell culture, allowed to reach confluence, and subcultured . The freshly isolated or subcultured cells were loaded into diffusion chambers and implanted into the peritoneal cavity of athymic mice to test their osteo-chondrogenic potential in a contained in vivo location . Freshly isolated periosteal cells formed both bone and cartilage tissue in such test chambers, but with a relatively low incidence. In contrast, cultured periosteal cells consistently gave rise to bone and cartilage even after 10 population doublings . With further passages of cells, the osteo-chondrogenic potential diminished substantially, until complete loss of expressivity at 16 population doublings or longer . Cultured muscle fibroblasts, when loaded into diffusion chambers under identical conditions to those of cultured periosteal cells, formed neither bone nor cartilage . These observations suggest that periosteal cells of young chicks contain subsets of progenitor cells or mesenchymal stem cells which possess the potential to differentiate into osteoblasts or chondrocytes, and this potential is retained after enzymatic isolation and for several population doublings in culture. Key Words : Periosteal-derived cells-Mesenchymal cellsCell culture-Diffusion chamber-Bone-Cartilage .

Introduction Evidence is available to support the hypothesis that there is a population of mesenchymal progenitor, or stem cells, present in bone marrow and periosteum which is capable of differentiating into several different tissues including bone and cartilage (Friedenstein et al . 1970 ; Friedenstein 1976 ; Ashton et al . 1980 ; Owen and Friedenstein 1988) . With regard to bone marrow, Friedenstein et al . (1970) were the first investigators who observed bone formation by bone marrow cells loaded into diffusion chambers and hypothesized the presence of determined osteogenic pre181

1 82 and/or cartilage tissue in diffusion chambers and, if so, to establish whether and when, in the process of subculturing, these periosteal cells lose this osteo-chondrogenic potential . Materials and Methods Periosteal cells

Tibial periostea of one-week-old White Leghorn chicks were used as the source of periosteal cells . The methods of periosteal cell preparation were described in detail elsewhere . (Nakahara et al . in press) . Briefly, overlying muscles and fasciae on the antero-medial surface of tibiae were carefully dissected away . The periostea were then digested with collagenase-trypsin and the liberated cells were counted after exclusion of nonviable cells with trypan blue (0 .4% ; Gibco Laboratories, NY) and seeded at 1 .0 x 10 6 per 100 mm plastic tissue culture dish in 8 mL of modified Ham's F-12 Medium (Gibco Laboratories, NY) supplemented with selected lots of 10% fetal bovine serum (J . R . Scientific Inc ., Woodland, CA) and antibiotics (Whittaker Bioproducts Inc ., Biggs Ford Road, MD) . Aliquots of freshly isolated cells were also loaded into diffusion chambers as described below . The plated cells were cultured at 37°C in 95% humidified air plus 5% CO, and the medium was replaced every three days . Subculture protocol

After 10-12 days of primary culture, when the cells reached confluence, they were harvested by treatment with trypsin-EDTA (0 .25% trypsin-1 mM EDTA) for 5-10 min, counted in a hemocytometer, and split 1 :4 for replating . The number of cells in a confluent 100 mm dish was 1 .3-1 .6 x 107 ; thus, 3 .25-4 .0 x 106 cells were seeded into a 100 mm dish upon replating . Subculture was continued in the same way with 1 :4 dilution upon replating every 7-9 days when the cells reached confluence .

H . Nakahara et al : Osteo-chondrogenic periosteal-derived cells against type II collagen (116B3 ; Linsenmayer and Hendrik 1980) . Growth curves of cells

To assess the dynamic mitotic events, subcultured cells at various passages were seeded into 35 mm dishes at 1 .0 x 10 5 cells per dish . Cells were detached by treatment with trypsin-EDTA and counted in a hemocytometer at various times starting 24 h after seeding . Cell numbers in three different dishes were counted at each time point and the means were plotted on a log scale against time on a linear scale ; these data are presented in Figure 4 . Bone specific markers Two additional monoclonal antibodies were used in order

to show that osteogenesis had occurred in diffusion chambers . The methods of generation of these antibodies are briefly described below . SH-1 (bone Gla protein specific probe) . SH-1 antibody generation : The SH-1 monoclonal anti-

body to chick bone Gla protein (BGP) was generated during the development of monoclonal antibodies against bovine noncollagenous proteins with chondrogenic stimulating activity (Syftestad et al . 1985) . Mice were immunized with a bovine bone extract which was partially purified for chondrogenic stimulating activity but contained BGP as a contaminant . Bone matrix extract was prepared from long-bone diaphyseal shafts of 1-year-old steer and fractionated by DEAE chromatography and Concanavalin A Sepharose chromatography as described elsewhere (Syftestad et al . 1985) . A 7-month-old male Balb C mouse was immunized with 2 mg of antigen dissolved in . 1 2 mL Complete Freund's Adjuvant intraperitoneally . Booster injections were given in Incomplete Freund's Adjuvant at 3, 6, and 9 weeks . In addition, the 4th and 5th boosters were given in the 9th week on consecutive days just prior to the fusion . The fusion was performed as previously described (Bruder and Caplan 1989a) .

Muscle fibroblasts

Fibroblasts were obtained from the same animals from which the periosteal cells were prepared . The methods were also described in detail elsewhere (Nakahara et al . in press) . Briefly, minced thigh muscles were placed onto tissue culture dishes in Eagle's Minimum Essential Medium (Gibco Laboratories, NY) supplemented with 10% fetal bovine serum and antibiotics . After 7 days, cellular outgrowths were harvested by trypsin-EDTA treatment and replated at 2 .0 x 106 cells per 100 mm culture dish for the first subculture . The medium was replaced every day . Cells reached confluence 6-8 days after replating and were split 1 :4 for the second subculture and plated into 100 mm dishes .

Hybridoma screening and cloning : Culture supernatants of hybridoma colonies were screened by ELISA against the immunizing antigen which was coated onto 96well plastic plates . Hybridoma colonies which tested positive were cloned twice by limiting dilution . After colony growth, culture supernatants were retested for positive reactivity against the original immunogen . The supernatants of cloned hybridoma colonies were then screened by ELISA against purified chick BGP (generously provided by Dr . Peter Hauschka, Children's Hospital Medical Center, Boston, MA) coated onto 96-well plastic plates . Three antibodies were reactive against the chick BGP on ELISA . One of these, SH-1, was further characterized .

Evaluation of osteogenic or chondrogenic phenotype in vitro

ting : Unfractionated bovine bone extract was loaded onto

Upon the first subculturing, some periosteal cells or muscle fibroblasts were seeded into 35 mm dishes at 1 .0 x 105 per dish . Two days after the cells reached confluence, they were evaluated immunocytochemically with monoclonal antibodies against osteogenic cell surface antigens (SB-1, -2, and -3 ; Bruder and Caplan 1989a, 1989b) or

SDS polyacrylamide electrophoresis and immunoblot-

two lanes and purified chick BGP was loaded onto one lane of a 5-17 .5% SDS polyacrylamide gel . The gel was prepared and run using the discontinuous buffer system of Laemmli (1970) . When the dyefront reached the bottom of the gel, electrophoresis was stopped and the proteins in the gel were electrotransferred (Towbin et al . 1979) to nitrocellulose (BioRad, Richmond, CA) . The nitrocellulose was

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cut into strips corresponding to lanes of the electrophoretic gel . One strip containing electrophoretically separated bovine bone proteins was stained with amido black to visualize the protein pattern . An identical strip and the strip containing chick BGP were blocked with 5% bovine serum albumin in PBS (blocking buffer) and then incubated with SH-1 hybridoma culture supernatant at 1 :5000 dilution in blocking buffer . Nitrocellulose strips were rinsed in blocking buffer and incubated with alkaline phosphataseconjugated goat anti-mouse IgG (Cappel, Rockville, MD) . Strips were rinsed in blocking buffer and incubated in alkaline phosphatase substrate (NBT, BCIP) by Promega (Fisher, Pittsburgh, PA) . The enzymatic reaction responsible for visualization was stopped by rinsing strips with distilled water . SB-5 (osteocyte specific probe) .

Fusion and hybridoma production : The cell fusion, hybridoma selection, and cloning procedures for the generation of monoclonal antibodies against osteogenic cells have been detailed elsewhere (Broiler and Caplan 1989a, 1989b) . The generation and detailed analysis of monoclonal antibody SB-5, which reacts with an epitope on the surface of osteocytes, is the subject of another report (Broiler and Caplan 1990) and will not be detailed here . Following hybridoma production and screening protocols which included tissue section immunocytochemistry as described below, the SB-5 cell line was cloned by limiting dilution several times, expanded into large scale culture, grown as ascites tumors, and frozen in liquid nitrogen . Tissue section immunohistochemistry : The antigenic determinant recognized by SB-5 was proven to be extremely sensitive to fixation, which limited the possibilities of tissue processing procedures . As such, tissues were dissected in phosphate buffered saline (PBS), rinsed repeatedly in 5% sucrose-PBS, placed in Tissue-Tek, O .C .T . embedding medium (Miles Laboratories, Inc ., Naperville, IL), frozen in liquid nitrogen, and stored at -20°C . Frozen sections (6-p.m-thick) were cut from calvaria of 16-day chick embryos at -15°C with an I .E .C . model Minitome/ microtome cryostat, placed on albumin-coated slides, and air-dried . All sections were stored in tight boxes at -20°C until used . Tissue sections were incubated with primary antibody for I h at 25°C in a high humidity chamber . Culture supernatant was used undiluted, while ascites fluid was diluted 1 :100 in PBS with 1% bovine serum albumin (BSA) (BPBS) . Following this incubation, the slides were rinsed three times with B-PBS and then incubated for 1 h with fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin (FITC-GAMIg) secondary antibody (Organon Teknika Corp ., West Chester, PA) diluted 1 :100 in B-PBS . The sections were again repeatedly rinsed with B-PBS, and coverslipped in a medium containing glycerol :PBS (9 :1) pH 8 .5 and 0 .01 M p-phenylenediamine (PPD) (Eastman Kodak, Rochester, NY) to retard photobleaching (Johnson et al . 1982) . Immunostained slides were then examined and photographed with an Olympus BH-2 epifluorescent/phase photomicroscope . Control experiments consisted of tissue sections that were incubated with nonimmune mouse serum, culture supernatant from SP2/0-Ag-14 cells, or B-PBS, followed by FITC-GAMIg secondary antibody as described above . None of these

control tissue sections displayed a selective immunofluorescent staining pattern . Diffusion chambers

Diffusion chambers were made from a plastic ring (2-mmthick, 9 mm i .d . ; Millipore Corporation, Bedford, MA) bounded by two microporous cellulose acetate and nitrate membranes (HAWP 01300 ; Millipore Corporation) of 100µm thickness and 0 .45 µm pore size (chamber volume 127 pi) . Freshly isolated periosteal cells or subcultured cells detached from the culture dishes by trypsin-EDTA treatment were rinsed two times with Tyrode's salt solution (T 2145, Sigma, MO) and finally resuspended in serum-free modified Ham's F-12 Medium or Eagle's Minimum Essential Medium for periosteal cells or muscle fibroblasts, respectively . The cells were then loaded into diffusion chambers (5 x 106 cells in 100 .pL per diffusion chamber) through a hole in the side of the ring, and the hole was then sealed with cement (XX 70 000 01, Millipore Corporation) . Host animals

Athymic mice (NIH strain, nu/nu, outbred, 6- to 8-weekold males) were used as host animals . Through a midline celiotomy, usually two diffusion chambers per mouse were implanted into the peritoneal cavity . Histologic examination of diffusion chambers

Four weeks following the implantation of diffusion chambers, they were recovered, fixed with 10% buffered formalin (pH 7 .0) and embedded in paraffin . Serial paraffin sections perpendicular to the plane of the filters were stained with toluidine blue, hematoxylin eosin, or von Kossa stain followed by counterstaining with hematoxylin eosin . Some of the recovered diffusion chambers were immunocytochemically assessed with the bone specific probes (SH-1 and SB-5) . Staining with SB-5 was conducted under the same protocol as for calvaria of 16-day embryos described above using frozen sections of diffusion chambers . For SH-1 staining, frozen sections perpendicular to the plane of the Millipore filters were demineralized with 4 .13% EDTA, pH 7 .4 (Warshawsky and Moore 1967) for 2 h at 4°C and then extensively washed with 5% sucrosePBS . They were then incubated for 1 h with SH-1, rinsed with B-PBS, and then reacted for an additional hour with FITC-GAMIg secondary antibody diluted 1 :100 in B-PBS . Incidence of bone and cartilage formation was expressed as the number of diffusion chambers with positive bone and cartilage formation per number of diffusion chambers recovered (Table I). Each of the subculture groups was derived from 17 separate periosteal cell preparations and the incidence shown in Table I is the summation of the results from those 17 separate experiments . Results Specificity of antibodies SH-I and SB-5

SH-l . Figure 1 shows an immunoblot analysis of the SH-1 monoclonal antibody to electrophoretically separated bovine bone proteins and purified chick bone Gla protein . The positive reactivity of SH-1 to bovine BGP (lane c) and

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Table 1 . Incidence` of bone and cartilage formation . Source of Cells Loaded into Diffusion Chambers Periosteal cells freshly isolated primary culture 1st subculture 2nd subculture 3rd subculture 4th subculture 5th subculture 6th subculture 7th subculture 10th subculture 12th subculture Muscle fibroblasts 1st subculture 2nd subculture

Approximate P.D .b at Loading into Diffusion Chambers

0 4 6 8 10 12 14 16 18 24 28

a b c d

Incidence of Bone and Cartilage Formation

317 9/9 10/10 12/12 12/12 4/10 1/8 0/9 0/10 0/8 0/10

(42 .9%) (100`70) (100%) (100%) (100%) (40%) (12 .5%) (0%) (0%) (0%) (0%)

0/7 018

(0%) (0%)

Incidence : number of diffusion chambers positive for bone and cartilage formation/number of diffusion chambers recovered at 4 weeks following implantation . b P.D . : population doublings . Ultimate passage numbers of subculture varied from 7 to 13 depending on cell preparation . The incidence shown in this Table is the summation of the results from 17 separate experiments .

not to the many other proteins in the crude bovine bone extract (lane b) illustrates the high degree of selectivity of the antibody for BGP over other bone proteins . In separate experiments, SH-1 reacted selectively to BGP in human and rat bone extracts (data not shown) . Crossreactivity of the monoclonal antibody to chick BGP is observed in lane d .

SB-5 . In frozen sections of embryonic stage 35 tibiae and developing calvaria, the only osteogenic cell type immunostained by SB-5 were the osteocytes . Newly synthesized bone matrix in some regions was also immunoreactive, but neither osteoblasts nor pre-osteoblasts were stained by SB-5 . Figure 2 illustrates the cell surface staining of osteocytes in a cryosection of a 16-day embryonic chick calvaria . Slender processes extending from the surface of osteocytes are also immunostained . These cytoplasmic extrusions make contact with neighboring cells via matrixfree canaliculi within the bone . A thorough presentation of the emergence of the SB-5 antigen during limb morphogenesis is the subject of another study (Bruder and Caplan 1990) . In summary, the immunoreactivity of SB-5 with osteogenic cells is limited to those cells possessing the morphologic features of osteocytes . In addition to the staining of osteogenic tissue by SB-5, immunoreactivity is present in cryosections of developing lung, feather, and kidney .

Fig . 1 . Immunoblot of the SH-I monoclonal antibody to crude bovine bone extract and purified chick BGP . Lanes : (a) molecular weight standards stained with amido black ; (b) crude bovine bone extract stained with amido black ; (c) crude bovine bone extract incubated with SH-1, then alkaline phosphatase-conjugated goat anti-mouse IgG and visualized by addition of alkaline phosphatase substrate ; (d) Purified chick BGP incubated with SH-1 and processed as in lane (c) . Molecular weight markers from top to bottom are 200,000, 116,250, 97,400, 66,200, 45,000, 31,000, 21,500, 14,400 (small arrows) . Large arrow represents the electrophoretic migration position of protein immunoreactive to the SH- I antibody.

matically released and replated at 1 :4 dilution ; those replated cells again showed fibroblast-like morphology (Fig . 3b) similar to muscle fibroblasts (Fig . 3c) but no longer showed colony-like groupings . Subculture was continued in the same way with 1 :4 dilution upon replating every 7 to 9 days when the cells reached confluence . During the subculture period, periosteal-derived cells looked regular in appearance and gave the impression of a relatively homogeneous population of fibroblast-like cells . Replated periosteal-derived cells did not overtly express either osteogenic or chondrogenic properties in monolayer culture as evidenced by immunocytochemical studies with osteoblast-specific probes (Bruder and Caplan 1989a, 1989b) or an antibody against type II collagen (Linsenmayer and Hendrik 1980) (data not shown) . Muscle fibroblasts also were not positive by these immunocytochemical criteria . Once past the 10th subculture (approximately 24 population doublings) of periosteal-derived cells, the plating efficiency diminished as did the rate of cell proliferation as judged by how many days were required to reach confluence (Fig . 4) . Eventually confluence could not be achieved at the 13th subculture (approximately 30 population doublings) . Diffusion chamber experiment

Cells in culture On day 1 of primary culture of enzymatically liberated periosteal cells, plating efficiency of the cells was observed to be 90-95% . These adherent cells were in a fibroblastlike morphology, and grew in discrete colonies (Fig . 3a). Growth potentials of each colony varied ; some colonies grew more rapidly than others, and the cells eventually reached confluence after 10-12 days of primary culture . When these cells reached confluence, the cells were enzy-

Four weeks following the implantation of diffusion chambers containing freshly isolated periosteal cells, some chambers (42 .9%) were positive for both bone and cartilage tissue with the bone tissue characteristically formed along the inner surface of the Millipore filters (Fig . 5a-d) . Mineralization of bone matrix was visualized by von Kossa staining (Fig . 5a) . Bone Gla protein was immunocytochemically detected in the mineralized bone matrix (Fig . 5b) . Immunoreactivity against the surface of cells which

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Fig . 2 . Expression of the SB-5 antigen in a frozen section of calvaria from a 16 day-old chicken embryo . Cryosections were stained with antibody SB-5, then FITC-GAMIg, and viewed with immunofluorescent (A) or phase contrast (B) optics . Cell surface immunostaining of numerous osteocytes trapped within bone matrix (b) is compared with the immunonegative pereosteal cells (p) and osteoblasts (ob) on the surface of bony trabeculae . Cell processes emerging from osteocytes are clearly immunostained (arrow) . x 512, were encased in this matrix was observed with SB-5 (Fig . 5c) . Cartilage tissue was usually found to be adjacent to the bone tissue toward the middle of the chambers (Fig . Sd) . Loose, fibrous connective tissue was always observed in the middle of the chambers which contained bone and cartilage (Fig . 5, a and d) . The remaining diffusion chambers which had been loaded with freshly isolated periosteal cells were negative for bone and cartilage formation and were filled with only loose, fibrous connective tissue . Several of these chambers were entirely serially sectioned to insure the absence of cartilage or bone . The incidence of bone and cartilage formation in diffusion chambers loaded with either freshly isolated cells or subcultured cells at various passages is shown in Table I . Diffusion chambers containing cultured periosteal-derived cells from up to the third subculture (approximately 10 population doublings) consistently gave rise to both bone and cartilage in the same manner as the positive chambers which were loaded with freshly isolated periosteal cells . With further subculturing, however, the incidence of bone and cartilage formation substantially decreased ; eventually bone and cartilage could not be detected when chambers were loaded with cells which had been subcultured for 16 population doublings (6th subculture) or longer . Only loose, fibrous tissue formation was observed in these cases (Fig . 6) . Neither bone nor cartilage formation was seen in diffusion chambers in which once or twice subcultured muscle fibroblasts had been loaded ; only loose, fibrous tissue formation was observed in these cases . This fibrous tissue looked similar to that formed in the middle of the chambers which contained bone and cartilage . Discussion Diffusion chambers implanted in vivo have been previously used to test phenotypic potential of mesenchymal cells from bone marrow or other tissues (Friedenstein et al . 1970 ; Owen and Friedenstein 1988) . The diffusion chamber assay tests the "intrinsic" phenotypic potential of loaded cells in an in vivo microenvironment without direct cell-tocell contact of host cells which are excluded by the inter-

vening Millipore filters . Using this assay system, Friedenstein and his colleagues have extensively studied potential phenotypic properties of the stromal reticular cells of hematopoietic and lymphoid organs (Friedenstein et al . 1970 ; Friedenstein 1976 ; Owen and Friedenstein 1988) . Their approach utilizes the technique of selective cloning under monolayer conditions to produce clones of fibroblast colony-forming cells . Cell cultures from bone marrow, spleen, thymus, and lymph nodes under these conditions yield fibroblast colony-forming cells in quantities unique to each tissue . While all of these cells form colonies of fibroblasts with similar morphology, their phenotypic properties, as tested by in vivo cultures in diffusion chambers, vary depending on the tissue origin . The fibroblast colonyforming cells from bone marrow, for example, can undergo osteogenesis in diffusion chambers, while those from spleen cannot . Thus, Friedenstein and coworkers suggested that fibroblast colony-forming cells from hematopoietic and lymphoid organs differ in their inherent phenotypic potential depending on the tissue origin . In the present study, liberated cells from periosteum also appeared fibroblast-like in monolayer cultures throughout the subculture period and they did not seem to possess overt osteogenic or chondrogenic properties in vitro as judged by their morphology and the lack of reactivity with probes to phenotype-specific antigens of osteoblasts or chondrocytes . Nevertheless, at up to 10 population doublings in culture, these cells consistently gave rise to bone and cartilage tissue in diffusion chambers . These observations suggest that enzymatically isolated, cultureselected and -expanded periosteal-derived cells have a dual potentiality to differentiate into osteoblasts or chondrocytes . Whether these cultured periosteal-derived cells contain several different cell subsets including committed osteoprogenitor cells and chondroprogenitor cells, or undifferentiated mesenchymal stem cells capable of differentiating into osteoblasts and chondrocytes, cannot be directly deduced from this study. Bone tissue was usually observed along the inner surface of the Millipore filter membranes, while cartilage tissue was observed to be more centrally located within the chambers . This localization of bone and cartilage tissue in



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2 1

0.3 12 4

e 12 Tim* ( days )

Is

Fig. 4 . Growth curves of periosteal cells (9 •) and muscle fibroblasts (e •) at various passages . S1,,,l,0,13 ; Ist, 4th, 7th, 10th, 13th subcultured cells . Muscle fibroblasts were tested only at first subculture . Each standard deviation of the means did not exceed 5% .

Fig . 3 . Morphological appearance of cells in culture . (a) Colonial growths of periosteal cells in primary culture (x 157) . (b) Once subcultured (S,) periosteal cells (x 157) . (c) Once subcultured (S,) muscle fibroblasts (x 157) . diffusion chambers coincides with that reported by Ashton et at . (1980) and Johnson et al . (1988) who also observed bone and cartilage formation when bone marrow cells were loaded into diffusion chambers . We suspect that locally produced cues control the phenotypic expression of loaded cells : next to the Millipore filter membrane, loaded periosteal-derived cells may be exposed to relatively high oxygen tension and nutrients brought by the host vasculature across the pore of the filter membrane . Under such conditions, embryonic chick mesenchymal cells have been reported to favor differentiation directly into osteoblasts (Basset 1962 ; Hall 1970 ; Osdoby and Caplan 1979) . In contrast, in the middle of the diffusion chambers, the cells are

under the influence of a lower nutrient accessibility and oxygen tension derived, again, from the host vasculature . Under such conditions chick mesenchymal cells have been reported to differentiate into chondrocytes (Basset 1962 ; Caplan 1970 ; Hall 1970 ; Caplan and Koutroupas 1973) . The incidence of bone and cartilage formation by freshly isolated periosteal cells was significantly lower than that by cultured periosteal-derived cells up to 10 population doublings . This may be due to the possible cellular damage by prolonged enzymatic digestion during liberation of periosteal cells . With subsequent subculturing past 10 population doublings, the cells seemed to quickly lose the potential to differentiate into osteoblasts or chondrocytes . In this regard, Simmons et al . (1982) assessed the osteogenie potential of cultured neonatal mouse calvarial cells using diffusion chambers, and reported loss of their osteogenie potential depending on length of time in culture . Moskalewski et al . (1983) reported that enzymatically isolated rat calvarial osteoblasts gave rise to bone tissue when inoculated intramuscularly ; in contrast, once these calvarial osteoblasts were introduced into cell culture for three population doublings or longer, they did not form bone as assayed in vivo . liken together, the loss of osteogenic potential with prolonged culture seems to occur regardless of the tissue origin of cells . This phenomenon may be attributable to the loss of inherent potential to differentiate into osteoblasts or chondrocytes due to the prolonged culture period and exposure to nonoptimal levels of growth factors in the culture medium . Indeed, once past the 10th subculture (24 population doublings) of periosteal-derived cells, they looked nonviable and eventually were unable to proliferate at the 13th subculture (approximately 30 popes lation doublings) . Another possible explanation for the loss of osteogenic potential by long-term subcultured periosteal-derived cells is that, during the subculture period, periosteal mesenchymal progenitor or stem cells respon-

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Fig . 6. Loose, fibrous tissue (F) formation in a diffusion chamber loaded with seventh subcultured periosteal cells (M : filter membrane, hematoxylin-eosin staining, x 308), sible for bone or cartilage formation may have been overgrown by some other cell subsets such as nonstem cell fibroblasts . Importantly, these studies raise the possibility that, like bone marrow, the periosteum contains subsets of mesenchymal progenitor or stem cells which are capable of differentiating into specific phenotypes depending on local cues, and this potential can be retained in cell culture for periods of up to several population doublings .

We thank Dr . D . Lennon for his assistance in cell culture, Ms . S . Miller for her assistance in histologic preparations, and Ms . E . Zborowska (Cancer Research Center, Case Western Reserve University) for taking care of the athymic mice . We also thank Dr . T . Linsenmayer (Tufts University, Boston) for his generous gift of anti-type II collagen antibody . Scott P. Bruder was supported by the Arthritis Foundation and a Medical Scientist Training Grant GMO-7250-13 . This research was supported by grants from the National Institutes of Health . Acknowledgments :

References Ashton, B .A . ; Allen, T. D . ; Howlett, C . R . ; Eaglesom, C. C . : Hattori . A . ; Owen, M . Formation of bone and cartilage by marrow stromal cells in diffusion chambers in viva . Clin . Orthop . 151 :294-307 ; 1980. Bab, I . ; Passi-Even, L. . ; Gazit, D . ; Sekeles, E . ; Ashton, B . A . ; PeylanRamu . N . ; Ziv, L'.. Ulmansky, M . Osteogenesis in in viva diffusion chamber cultures of human marrow cells . Bone and Mineral 4:373-396; 1988 . Bassett, C . A . L . Current concepts of bone formation . J . Bone Joint Sure. 44-A :1217-1244 ;1962 .

Fig. 5 . Tissues formed in a diffusion chamber loaded with freshly isolated periosteal cells . (a) Bone (B) and cartilage (C) formation 4 weeks after introduction into the peritonea] cavity. Mineralization

of bone matrix can be detected by von Kossa stain (M : filter membrane of a Millipore filter ; F : loose, fibrous tissue- von Kossa stain followed by counterstaining with hematoxylin-eosin staining, x 308) . (b) Bone Gla protein is immunocytochemically detected with SH-I in the mineralized matrix (X308) . (c) Surface of the cells which are trapped within the mineralized matrix is immunostained with SB-5 . Serial section to that shown in Fig . 5b . X 308 . (d) Cartilage (C) formation adjacent to bone (B) toward the middle of the diffusion chamber can be detected by toluidine blue staining (M: Millipore filter membrane ; F : loose, fibrous tissue .) Serial section to that shown in Fig . 5a . x 308 .

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