Chondrogenesis and osteogenesis of bone marrow-derived cells by bone-inductive factor

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Chondrogenesis and Osteogenesis of Bone Marrow-derived Cells by Bone-inductive Factor K . HARADA, 1 S . OIDA? and S . SASAKI2

I 2

The .Second Department of Oral Surgery, end

Department of Biochemistry, Faculty of Dentistry, Tokyo Medical and Dental University, 545, Yushima, 1-Chome, Bunkyo-ku,

Tokyo 113, Japan . Address for correspondence and reprints : Dr. K . Harada, Second Department of Oral Surgery, Tokyo Medical and Dental University, 5-45, Yushima, 1-Chome . Bunkyo-ku, Tokyo 113, Japan . Abstract

Reddi, 1985) . The cells derived from muscle which cannot differentiate to form bone and cartilage without osteo-inductive factors have been termed "inducible osteogenic progenitor cells" (IOPC) (Friedenstein, 1976) . On the other hand, it is known that when cells obtained from the bone marrow are transplanted to heterotopic sites or implanted into an animal within a diffusion chamber, they differentiate to form bone or cartilage (Friedenstein et al ., 1966, 1968 ; Ashton et al ., 1980 ; Budenz et al ., 1980 ; Bab et al ., 1986) . These cells have been designated "determined osteogenic progenitor cells" (DOPC) (Friedenstein . 1976) . However, the presence of IOPC in bone marrow cells also has been postulated (Friedenstein . 1976) . In this study, we determined the effects of a decalcified bone matrix on the ability of bone marrow cells to differentiate to form cartilage and bone within a diffusion chamber . In addition, reconstituted pellets of a 4 M guanidine-extracted bone matrix (G-res) with partially purified BMP were implanted subcutaneously or placed in a diffusion chamber with bone marrow cells . Furthermore, marrow cells were cultured in vitro for 10 days and proliferated cells were inoculated into a chamber. The possibility of the presence of IOPC in a marrow-derived fibroblast-like cell preparation was also investigated .

Rat bone marrow cells were intraperitoneally implanted within a diffusion chamber with a decalcified bone matrix or a 4 M guanidine hydrochloride extracted matrix (G-res) as control . The chamber was harvested after 28 days and soft X-ray photography, histological examination, determination of alkaline phosphatase activity and calcium content were performed . With the decalcified bone matrix, cartilage and bone formation was observed and both alkaline phosphatase activity and calcium content were significantly higher than those in control chambers . Each chromatographic fraction on Sephacryl S-200 of the 4 M guanidine hydrochloride extract (G-ext) from the decalcified bone matrix was reconstituted with G-res and implanted either subcutaneously or intraperitoneally within a diffusion chamber with marrow cells . Intrachamber or subcutaneous cartilage and bone formation was detected by only one chromatographic fraction . When marrow-derived fiibroblast-like cells were implanted intraperitoneally within a diffusion chamber with a decalcified bone matrix, cartilage and bone formation was detected, which was not the case with G-res . These results suggest that a certain factor, probably bone morphogenetic protein, which induces ectopic bone formation, allows marrow cells to differentiate into bone and cartilage tissues and there may exist so-called "inducible osteoprogenitor cells" in the marrow-derived fibroblast-like cell preparation . Key Words : Chondrogenesis-Osteogenesis-Bone Marrow-Diffusion chamber-BMP.

Introduction A decalcified bone matrix, when implanted into various extraskeletal sites . induces the differentiation of cartilage and bone in the host tissues (Urist, 1965 ; Urist et at., 1969) . The factor responsible for this induction of osseous tissues was found to be a protein having an approximate molecular weight of 18 K and has been termed "bone morphogenetie protein" (BMP) (Urist et al ., 1984) . The cells which respond to BMP in changing their phenotype are helieved to be undifferentiated mesenchymal cells derived from muscle or muscle fascia (Nogami and Urist . 1970 . 1974 ; Nakagawa and Urist, 1977 ; Sampath et al ., 1984 :

Fig 1 . Soft X-ray radiographs of two diffusion chambers 28 days after int aperitoneal implantation . (a) Chamber inoculated with bone marrow cells and decalcified bone matrix . (b) Chamber inoculated with hone marrow cells and G-res . Radiopaque contiguration which indicates a calcified structure is observed only in (a) .

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K . Harada et al . : Chondrogenesis and ostcogenesis of bone marrow

a

c

F

Fig . 2. Photomicrographs of histological sections of the specimens in the diffusion chambers as shown in Figure I . (a) Bone (B) and cartilage (C) formation in the diffusion chamber inoculated with marrow cells and decalcified bone matrix (M) (Von Kossa and toluidine blue staining) . (b) The same section stained by the azo dye method . Newly formed cartilage (C) exhibits alkaline phosphatase activity . (M) : decalcified bone matrix . (c) Only loose connective tissue is observcd in the diffusion chamber inoculated with marrow cells and G-res (R) (toluidine blue staining) . (F) : Millipore filter (x 110) .

Materials and Methods Preparation of bone rnatrix The diaphyses of long bones from male Wistar strain rats (about 300 g body weight) were scraped clean of soft tissues and marrow, frozen in liquid nitrogen, and pulverized with a stainless steel mortar and pestle . The powdered bone was strained through a mesh of 74-420 .pm and the fine bone powder was decalcified with 0 .6 N HCI for 16 h at 4°C and excess acid was washed out exhaustively with

distilled water up to pH 6, followed by successive washing with ethanol and chloroform-methanol (1 :1) . The resultant particles were dried at room temperature and stored at - 20°C . Guanidine extraction fhe decalcified bone particles were extracted with a 4 M guanidine hydrochloride (Gdn • HCI) for 16 h at 4°C and centrifuged for 30 min at 30,000 x g . The supernatant was dialyzed against distilled water and lyophilized (G-ext) .



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K . Harada et al . : Chondrogenesis and osteogenesis of bone marrow Al-p

activity & Ca content in diffusion chambers

xI0 - ' 4 .0

300

° 0 200

3

100

r

•4ree

mmau SI only

Fig . 5 . Soft X-ray radiographs of subcutaneously implanted pellets reconstituted with the chromatographic fractions in Figure 4 (top) and intraperitoneally implanted diffusion chambers inoculated with the pellets and marrow cells (bottom) . Only with fraction 111 (111, top and bottom) are radiopaque configurations due to calcified tissue observed .

04MI1 ednw

Fig . 3 . Alkaline phosphatase activity (hatched bars) and calcium content (open bars) in diffusion chambers inoculated with marrow cells and decalcified bone matrix (- demineralized bone), marrow cells and G-res (+ G-res), or marrow cells only (marrow cell only) . The far right bars represent the enzyme activity and calcium content in the freshly isolated bone marrow (freshly isolated) . Data represent the mean of four values : bars, SD .

distilled water and lyophilized . Each fraction (1 .5 mg) was reconstituted with 15 mg of G-res according to the method of Sampath et al . (1981) and the resultant pellets were dried at 37°C and stored at -20°C . Preparation

The insoluble residue of the bone matrix in 4 M Gdn • HCI was washed with distilled water and lyophilized (G-res) . Gel filtration chromatography and reconstitution G-ext was subjected to gel filtration through a precalibrated Sephacryl S-200 column (2 .6 x 94 cm) using 4 M Gdn • HCI/50 mM Tris - HCI (pH 7 .4), and 5-ml fractions were collected at a flow rate of 30 ml/h . The absorbance of the eluants was monitored at 230 nm and appropriate fractions were pooled into six fractions (I -- VI) . From 10 g of decalcified bone particles, 350 mg of fraction I, 68 mg of fraction 11, 32 mg of fraction III, 1 I mg of fraction IV, 3 mg of fraction V and I mg of fraction VI were recovered respectively . Fractions II, III and IV were dialyzed against

saphacrvl Rat G-ext

S-200 4M Gdn-HCI 50mM

Tris-HCI

off 7 .4

of marrow cell

suspension

Male Wistar strain rats (8 weeks old) were sacrificed and the femoral midshaft marrow was flushed out into Hanks' balanced salt solution with the aid of a syringe . A singlecell suspension of the marrow was obtained by pumping the tissue repeatedly through hypodermic needles of decreasing gauge . In vitro culture A tissue culture dish (100 mm in diameter) containing 10 ml of a-MEM supplemented with 10% fetal calf serum, 100 units of penicillin per ml, and 100 .g p of streptomycin per ml, was inoculated with I to 2 x 10' marrow cells . The medium was completely changed twice a week . After 10 days of culture, the confluent fibroblast-like cell layer was harvested by treatment with a solution of trypsin-EDTA in a serum free medium, and inoculated into a diffusion chamber . The number of viable cells was determined previously . Intraperitoneal implantation within a diffusion chamber

n, F. No

Fig . 4 . Sephacryl S-200 chromatographic profile of a 4 M guanidine hydrochloride extract from decalcified rat bone . Fractions 11 . III and IV were pooled, dialyzed, lyophilized, and reconstituted with G-res . Molecular weight standards were eluted at a (chymotrypsinogen A, M .W. 25000) and b (cytochrome C, M .W. 12500) .

Diffusion chambers were constructed of a lucite ring (2 mm thick with a center hole 12 mm in diameter) hound with filters of 0 .45 µm pore size (Millipore Corp .) . After sterilization with ethylene oxide gas, the chambers were inoculated with 2 .7 x 10' marrow cells or 3 .0 x 106 cultured fibroblast-likc cells . At the same time, 15 mg of decalcified bone particles, G-res or reconstituted pellets were loaded into the chamber . The chamber kept in Hanks' balanced salt solution was implanted intraperitoneally into a host rat (Wistar strain male, 8 weeks old) and harvested after 28 days .

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K . Harada ct al . : Chondrogenesis and osteogenesis of bone marrow

K . Harada et a1 . : Chondrogenesis and osteogenesis of bone marrow

Examination after implantation The chamber was radiographed with soft X-ray for 3 min at 20 KV and 3 mA . and its radiolucency was determined . Then the chamber was embedded in an embedding material (O .C .T . compound, Lab-Tee Products) and frozen at -80°C . Four-micron sections were cut with a cryostat and stained for alkaline phosphatase activity by the azo dye method . Some of the chambers were fixed in formalin and embedded in paraffin . Six-micron sections were stained with hematoxylin-eosin or toluidine blue . Additionally, sections were stained by the Von Kossa method, and counterstained with toluidine blue . Alkaline phosphatase activity in the diffusion chamber was determined by p-nitrophenyl phosphate as a substrate at pH 10 .0 and calcium content by spectrophotometry using orthocresolphthalein complexon (OCPC) .

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implanted chamber showed radiopaque configuration inside the lucite ring . Histologically, only the subcutaneously implanted pellet of fraction HI showed bone and bone marrow formation (Fig . 6b) . In accordance with this result, the marrow cells implanted within a diffusion chamber with the pellet containing fraction III differentiated to produce cartilage and bone (Fig . 6e). With fractions Ii and IV, no sign of bone or cartilage formation was detected in the subcutaneously implanted pellet or within the diffusion chamber (Fig . 6a,c,dj) . When marrow cells were cultured for 10 days in vitro, proliferation of fibroblast-like cells was observed (Fig . 7). Then, the confluent fibroblast-like cells were collected and implanted within a diffusion chamber with a decalcified bone matrix, and newly formed cartilage and bone were detected (Fig . 8) . Discussion

Results When freshly isolated bone marrow cells inoculated along with a decalcified bone matrix in a diffusion chamber were implanted into the peritoneal cavity of an allogeneic host for 28 days, a radiopaque configuration inside the lucite ring was observed (Fig . 1a) . On the other hand, when marrow cells were implanted along with a 4-M Gdn • HCIextracted bone matrix, the material in the chamber remained radiolucent (Fig . lb) . Histologically, marrow cells implanted along with a decalcified hone matrix were found to differentiate to produce cartilage, bone and fibrous tissue within the chamber (Fig . 2a) . In the frozen sections, newly formed cartilage was stained by azo dye ; thus it was found that the induced cartilage was expressing alkaline phosphatase activity (Fig . 2b). Marrow cells cultured with G-res produced only loose fibrous tissue (Fig . 2c) . In order to quantitate the difference in histological findings, alkaline phosphatase activity and calcium concentration in the specimens within the diffusion chamber were analyzed . The results are shown in Figure 3 . Both alkaline phosphatase activity and calcium content were significantly higher (p < 0.001) in the chamber inoculated with marrow cells and decalcified bone matrix than in those containing marrow cells and G-res or marrow cells alone- Alkaline phosphatase activity and calcium content in diffusion chambers loaded with marrow cells and G-res or marrow cells alone were not significantly different . These data suggest that the effects on bone marrow cells are principally due to the G-ext fraction derived from the decalcified bone matrix . Therefore . G-ext was fractionated by Sephacryl 5-200 column chromatography and the elution pattern is shown in Figure 4 . Fractions II, III and IV were reconstituted with G-res and implanted subcutaneously or intraperitoneally within a diffusion chamber containing bone marrow cells . Figure 5 shows the soft X-ray radiographs of the samples . Only with fraction III was the subcutaneously implanted pellet radiopaque and also the intraperitoneally

It is well known that two main cellular systems, the hemopoietic and stromal cell systems, comprise bone marrow . The stromal tissue is presumed to be an important part of the environment which influences proliferation and differentiation of hemopoietic cells (Owen, 1986) . Stromxl cells include cells classified under a variety of names such as mesenchymal cells, fibroblasts, reticular cells, preosteoblasts, or endothelial and adventitial cells associated with blood vessel walls (Ashton et al ., 1980 ; Owen, 1986) . When marrow cells are cultured in vitro, proliferation of fibroblast-like cells is usually observed . Friedenstein and his colleagues (1970) described these fibroblast-like cells as "fibroblastic colony forming cells" (FCFC, since they form colonies in monolayer culture . The presence of socalled "determined osteoprogenitor cells" (DOPC) in the FCFC preparation has been suggested because they differentiate to form cartilage and bone when inoculated under the renal capsule (Friedenstein et al ., 1968) or implanted within a diffusion chamber (Ashton et al ., 1980) . In our present study, when the chambers were inoculated with bone marrow cells (or marrow-derived fibroblast-like cells) alone, the frequency of bone and cartilage formation was extremely low. However, when the chambers were inoculated with bone marrow cells (or marrow-derived fibroblast-like cells) and decalcified bone matrix, bone and cartilage formation was observed in almost all implanted chambers . And subcutaneous or intra-chamber bone and cartilage formation was found only in fraction III after fractionation of G-ext with a Sephacryl S-200 column . This result suggests that a certain factor or factors in this fraction, which induce(s) ectopic bone formation, has (have) some effects on so-called "inducible osteoprogenitor cells" (IOPC) in the bone marrow as well as in our preparation of marrow-derived fibroblast-like cells . There is another possibility that some factor or factors in the bone matrix stimulated proliferation of DOPC in the bone marrow or fibroblast-like cells . However, when those fibroblast-like cells were cultured in vitro with G-ext (200 p .glml of medium) .

Fig . 6 . Photomicrographs of histological sections of implanted pellets reconstituted with chromatographic fractions in Figure 4 (top : a,h,c) and diffusion chambers inoculated with the pellets and marrow cells (bottom : d,c .f) . (a,d) : pellet reconstituted with fraction 11 - (b,e) : pellet reconstituted with fraction Ill - (c,fl : pellet reconstituted with fraction IV . In the subcutaneously implanted pellet with fraction III (h), the implanted bone matrix has disappeared and is replaced by newly formed bone (B) . When implanted in a diffusion chamber with fraction III (e), the bone matrix (M) is still present and newly formed bone (B) and cartilage (C) are observed . With fractions II and IV, only fibrous tissue is observed both in subcutaneously implanted pellets and within the diffusion chambers . (F) : Millipore filter . (H .E . staining . a .b .c,e x 110 ; d,f x52)

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K . Harada et al . : Chondrogenesis and osteogenesis of hone marrow phogenetic protein (BMP) having a molecular weight of 18K (Sampath and Reddi, 1984 ; Urist et al ., 1984) . Recently, Seyedin and his colleagues (1983, 1985) obtained two cartilage-inducing factors (CIF) from decalcified bone matrix and one of them was identified as transforming growth factor-p (TGF-p) (Ellingsworth et al ., 1986 ; Seyedin et al . . 1986) . In our present study, the molecular range of the fraction which contained bone-inducing activity, Sephacryl S-200 Fr . III, was between 14,000 and 30,000 . The molecular weight of TGF-{3 has been reported to be 26,000 (Seyedin et al ., 1985 ; Ellingsworth et al ., 1986) ; therefore, there is the possibility that the present activity responsible for bone and cartilage formation within diffusion chambers is due to either "bone morphogenetic protein" or TGF-p .

Acknowledgments : We thank Professor Syoji Enomoto for his helpful discussion, and Dr. Izumi Asahina for providing the chromatographic fraction on Sephacryl 5-200 .

References Fig . 7. Phase contrast micrograph of fibroblast-like cells derived from the bone marrow in monolayer culture . (x 150)

suppression of cell proliferation was observed (data not shown) . Green et al . (1984) also reported no significant increase in DNA content in the chamber containing marrow cells with or without hone matrix and concluded that the matrix was not responsible for the overall level of cell proliferation . This also supports the explanation that IOPC in marrow-derived fibroblast-like cells differentiated to bone and cartilage under the influence of osteoinductive factors . However, it cannot be completely ruled out that some bone-derived factor or factors bring(s) proliferative effects on DOPC at the expense of other cells within the diffusion chamber. A bone-inductive substance has been extracted and partially purified from bone matrix and designated bone mor-

Fig. 8 . Photomicrograph of histological section of a peritoneally implanted diffusion chamber inoculated with marrow-derived fibroblast-like cells and decalcified bone matrix. Formation of new bone (B) and cartilage (C) is observed . (M) : decalcified hone matrix . (F) : Millipore filter. (toluidine blue staining x I]0)

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Received : August 18, 1987 Revised : November 9, 1987 Accepted: November 11, 1987