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Endocrinology Vol. 142, No. 5 2087-2094
Copyright © 2001 by The Endocrine Society

ARTICLES

Effects of Cartilage-Derived Morphogenetic Proteins and Osteogenic Protein-1 on Osteochondrogenic Differentiation of Periosteum-Derived Cells1

Reinhard Gruber2, Christian Mayer2, Klaus Bobacz, Maria-Theresa Krauth, Winfried Graninger, Frank P. Luyten and Ludwig Erlacher

Clinic of Internal Medicine III (R.G., C.M., K.B., M.-T.K., W.F., L.E.), Department of Rheumatology, Vienna A-1090, Austria; School of Dentistry (R.G.), Department of Oral Surgery, University of Vienna, Vienna A-1090, Austria; and Division of Rheumatology (F.P.L.), University Hospitals, KULeuven, Leuven B-3000, Belgium

Address all correspondence and requests for reprints to: Ludwig Erlacher M.D., Clinic of Internal Medicine III, Department of Rheumatology, Allgemeines Krankenhaus, Waehringer Guertel 18–20, Vienna A-1090, Austria. E-mail: ludwig.erlacher{at}univie.ac.at


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Localization studies and genetic evidence have implicated cartilage-derived morphogenetic proteins-1, -2 (CDMP-1 and CDMP-2), and osteogenic protein-1 (OP-1) in the osteochondrogenic differentiation of mesenchymal progenitor cells during embryonic development and in postnatal life. Based on their expression pattern and the evidence that periosteum contains mesenchymal cells in the cambium layer that can undergo bone and cartilage formation, we hypothesized that CDMPs and OP-1 may be involved in long bone development and fracture healing. To test this hypothesis, periosteum-derived cells from young calves were cultured as monolayers under serum-free conditions with and without the addition of recombinant CDMP-1, CDMP-2 and OP-1. Phenotypic analysis indicate that periosteum-derived cell populations prepared, expanded, and cultured under the conditions described below, constitutively express messenger RNAs for the bone markers osteocalcin, osteopontin and collagen type I, and the chondrogenic markers collagen type II and aggrecan as determined by RT-PCR. Moreover, histologic examinations showed positive staining for alcian blue and alkaline phosphatase (AP). Treatment of periosteum-derived cells with CDMPs and OP-1 resulted in a dose-dependent increase of cell proliferation; CDMP-2 was less active in this regard. Furthermore, all growth factors enhanced osteogenic differentiation as assessed by a time- and dose-dependent stimulation of AP activity and OP-1 increased messenger RNA expression for osteocalcin and collagen type I. We further examined the effects of CDMPs and OP-1 on chondrogenic differentiation of periosteum-derived cells. Both CDMPs and OP-1 stimulated 35S-sulfate incorporation into newly synthesized macromolecules with OP-1 having a more pronounced stimulatory effect when compared with CDMP-1 and CDMP-2. Our results indicate that distinct members of the BMP-family increase the mitotic and metabolic activity of periosteum-derived cells. The enhancement of both the chondrogenic and osteogenic differentiation suggests that these growth factors might contribute to the local regulation of bone formation and fracture repair.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PERIOSTEUM is comprised of two tissue layers; the outer fibroblast layer provides attachment to tendons and ligaments, and the inner cambial region contains a pool of undifferentiated mesenchymal cells that support bone formation and repair (1). During embryonic and postnatal bone growth, mesenchymal progenitors differentiate directly into osteoblasts (2, 3), whereas during fracture healing these cells undergo both intramembranous ossification and chondrogenic differentiation with subsequent endochondral bone formation (4, 5). Furthermore, a number of studies have shown that when periosteum is autografted at heterotopic sites, either as free grafts or in diffusion chambers, mineralized and cartilaginous tissue could be detected (6). Based on these observations, the transplantation of periosteal tissue was used to repair articular cartilage defects in both animal models and humans in vivo (7, 8). Other investigators have also reported that culture-expanded cells, obtained by enzymatic digestion or outgrowth of periosteal explants, retain their osteochondrogenic potential (9, 10). Despite these observations, the molecular mechanism that is responsible for the proliferation and lineage-restricted differentiation of periosteal cells is largely unknown, though it has long been recognized that environmental factors including mechanical stimuli, oxygen tension, and growth factors play a role in this complex physiologic process.

Among the growth factors that target periosteal tissue, TGF-ß has been shown to stimulate the development of the chondrogenic phenotype and some authors suggest that it also inhibits the osteogenic pathway (11, 12, 13). Bone morphogenetic proteins (BMPs) belong to the same superfamily of structurally related proteins, but they are characterized by the unique ability to induce de novo bone and cartilage formation when implanted at ectopic sites (14, 15). BMPs are reported to initiate the migration and proliferation of mesenchymal progenitors, as well as their commitment into the chondrogenic and osteogenic lineage (16). Immunohistochemical studies have shown that BMP-2, -4, and OP-1 are expressed during the early stages of fracture repair in the periosteum, suggesting that BMPs may affect proliferation and differentiation of local progenitor cells (17, 18, 19). Evidence for this hypothesis came from Iwasaki and co-workers who reported that rhBMP-2 stimulates osteogenesis in ex vivo high-density cultures of chick periosteum-derived cells (20).

Cartilage-derived morphogenetic proteins-1, -2 (CDMP-1 and CDMP-2), also known as growth/differentiation factors 5 and 6 respectively, are closely related to OP-1 and form a subgroup within the BMP-family (21, 22). Genetic evidence suggests that their biologic function appears to be more restricted to cartilage tissue and joint formation because brachypodism mice and patients with chondrodysplasia, having a mutation in the CDMP-1 gene, show skeletal disorders like shortened bones of the limbs and abnormal joint development (23, 24, 25). Results from transgenic mice with targeted misexpression of CDMP-1 suggest that this growth factor enhances the commitment of mesenchymal cells into the chondrogenic lineage and their differentiation toward hypertrophy, thereby affecting endochondral ossification (26). Furthermore, CDMPs stimulate osteogenic differentiation of marrow stromal cells and enhance the metabolic activity of articular chondrocytes (27, 28). In addition, CDMP-1 has been shown to be expressed in a distraction osteogenesis (Ilizarov) model (29), as well as in explants of rabbit periosteal tissue (30). These observations led us to hypothesize that CDMPs may be involved in the regulation of osteochondrogenic differentiation of periosteal cells.

To test this hypothesis we used an in vitro model of bovine periosteum-derived cells (11) and investigated the ability of recombinant CDMP-1, CDMP-2 and OP-1 to regulate cell proliferation, osteochondrogenic differentiation, and cell metabolic activity. Our results showed that both CDMPs and OP-1 stimulate mitotic activity, the synthesis of cartilage matrix macromolecules, and the osteogenic differentiation. Furthermore, the data indicate that a proportion of cells within the monolayer cultures constitutively express a chondrogenic phenotype, independent of growth factor treatment.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Periosteum was obtained by aseptic techniques from anteromedial sites of the proximal tibia of 4- to 6-month-old calves. Areas of dissection were distant from cartilage tissue and perichondrium in the region of the epiphysis and articular surface. After mincing the tissue, cells were released by a 4-h digestion in 0.4% collagenase B (Roche Molecular Biochemicals, Mannheim, Germany), filtered through a cell strainer (Falcon, Becton Dickinson and Co. Labware, Lincoln Park, NJ) to remove debris and undissociated cell clusters. The cell filtrate was then centrifuged at 500x g for 10 min. Pellets were resuspended in DMEM and Ham’s F-12 (1:1; both from Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% FCS (PAA Laboratories, Linz, Austria) and 100 U/ml penicillin G, 100 µg/ml streptomycin, and 0.25 µg/ml Amphotericin (Life Technologies, Inc.). Cells were plated at a density of 1 x 104 cells/cm2 in 96- and 6-multiwell culture dishes or in chamber slides (Falcon). After culturing the periosteum-derived cells to 90% confluence, the growth medium was replaced by a chemically defined serum-free medium of Ham’s F12/DMEM (1:1) with ITS + culture supplement (Collaborative Biomedical Products, Bedford, MA), {alpha}-ketoglutarat (1 x 10-4 M), ceruloplasmin (0.25 U/ml), cholesterol (5 µg/ml), phosphatidylethanolamine (2 µg/ml), {alpha}-tocopherol acid succinate (9 x 10-7 M), reduced glutathione (10 µg/ml), taurine (1.25 µg/ml), triiodothyronine (1.6 x 10-9 M), hydrocortisone (1 x 10-9 M), PTH (5 x 10-10 M), ß glycerophosphate (1 x 10 mM), and L-ascorbic acid 2-sulfate (50 µg/ml) (Sigma, St. Louis, MO) (31). The cells were subsequently cultured with and without the addition of recombinant CDMP-1, CDMP-2, and human OP-1 at final concentrations of 10–300 ng/ml for 7 days at 37 C in 95% air and 5% CO2. The medium and the recombinant growth factors were replaced every other day.

Cell proliferation
The proliferation of periosteal fibroblasts cultured in 96-well plates (Packard, Meriden, CT) was measured by uptake of radiolabled [3H]-thymidine (0.5 µCi/well, Amersham Pharmacia Biotech, Buckinghamshire, UK). Subconfluent cells were washed, and preincubated with serum-free medium for 24 h. The growth factors were added for an additional 24 h and cells were pulsed with [3H]-thymidine for the last 6 h of culture. The cells were washed extensively with PBS and thymidine incorporation was measured directly in the culture plate by the addition of scintillant in a liquid scintillation counter (Packard, Meriden, CT).

Alkaline phosphatase assay
At the end of the indicated culture period histochemical staining of alkaline phosphatase (AP) was performed as described by D’Ippolito et al. (32) with minor modifications. Briefly, cells were fixed with neutral-buffered formaline for 20 min at 4 C, incubated with substrate solution at room temperature for 15 min, rinsed 5 times with distilled water, and photographed (Nikon F601, Tokyo, Japan). AP-substrate was prepared from Solution A (8 mg naphtol-AS-TR phosphate/300 µl N, N'- dimethylformamide; both from Sigma) and Solution B (24 mg fast blue BB salt/30 ml of 100 mM Tris-HCl; pH 9.6). Both solutions were mixed, 10 mg of MgCl2 was added, and used immediately after sterile filtration.

Enzymatic activity was determined in cell lysates that were solubilized with 0.1% Triton X-100. Aliquots (20 µl) of each sample were incubated with 100 µl AP substrate buffer (100 mM diethanolamine, 150 mM NaCl and 2 mM MgCl2, p-nitrophenylphosphate at 2.5 µg/ml) for 5–30 min at room temperature. Total cellular protein was determined using the bicinchoninic method (Pierce Chemical Co., Rockford, IL). AP activity is expressed as units per milligram protein with 1U defined as enzymatic activity that released 1 nmol p-nitrophenol/minute.

Macromolecule biosynthesis
On day 7 of cultivation, cells were metabolically radiolabled with 50 µCi/ml of [35S]-sulfate (carrier-free, Amersham Pharmacia Biotech) for 6 h at 37 C before they were harvested in guanidine-HCl buffer (4 M guanidine-HCl, 50 mM sodium acetate buffered at pH 7.2, in the presence of protease inhibitors). Unincorporated isotope was removed by using Sephadex G-25 (PD-10 columns; Pharmacia Biotech, Piscataway, NJ) columns. Total values were obtained by liquid scintillation counting of aliquots from void volume fractions and normalized to total cellular protein content.

RNA isolation and RT-PCR
Total cellular RNA was prepared from cultured cells before treatment and after 2-, 5-, and 7- days of growth factor treatment under serum-free conditions using the TRIzol reagent (Life Technologies, Inc.). Aliquots of 1 µg total RNA were digested with DNAseI as recommended by the manufacture (Life Technologies, Inc.), transcribed into complementary DNA (cDNA) (Amersham Pharmacia Biotech) using random hexamers primers and analyzed for the expression of aggrecan, collagen type II, osteocalcin, osteopontin, and collagen type I transcripts by PCR. Reactions were performed in an air thermal cycler (Idaho Technologies Inc., Idaho Falls, ID) with an initial denaturation at 94 C for 1 min, followed by 35 cycles of 94 C/1 sec -55 C/1 sec (62 C/1 sec for collagen type II) -72 C/40 sec, and an additional 2 min extension at 72 C. Amplification products were analyzed in 1.5% agarose gels, stained with Sybr-Green (Molecular Dynamics, Inc., Sunnyvale, CA), and photographed using a digital scanning system (Molecular Dynamics, Inc.). For bovine aggrecan, the primer set was: 5'-TCCCAGAATCCAGCGGTGAGAG (+6146-F), 5'-GCACAGGGCTTGAGGATTCG (+6592-R), for collagen type II (33) the primer set was: 5'-TCGGGGCTCCCCAGTCGCTGGTG (+152-F), 5'-GATGGAGAACCTGGTACCCCTGGA (+ 6661-R), for bovine collagen type I (34) the primer set was: 5'-GGAAGGAGAGAGCGGCAAC (+1165-F), 5'-ATACCAGGGAGACCCAC (+1522-R), for bovine osteopontin the primer set was: 5'-CCGAGGTGATAGTGTGGCTTAC (+516-F), 5'-TTCATATTGTCTCCCACCCTG (+979-R), for bovine osteocalcin the primer set was: 5'-GACAGACACACCATGAGAACC (+16-F), 5'-CTAGCTCGTCACAGTCAGGG (+277-R). In some reaction mixtures, reverse transcriptase was omitted to determine contaminating genomic DNA. For further controls, total RNA was used from bovine chondrocytes and from bovine synovia.

Northern blot analysis
Total RNA from day 7 cultures (10 µg/lane) was electrophoresed on a formaldehyde-containing agarose gel and transferred onto Nytran membranes (Schleicher & Schuell, Inc., Keene, NH). cDNA fragments of osteocalcin, collagen type I, and ß-actin were amplified by RT-PCR, purified, and labeled with 32P-desoxycytidine triphosphate (Amersham Pharmacia Biotech), using a random hexanucleotide-primed second-strand synthesis method (Amersham Pharmacia Biotech). After UV cross-linking, the membranes were prehybridized at 68 C for 30 min in hybridization buffer (Express Hyb; CLONTECH, Palo Alto, CA). Hybridization was carried out in the prehybridization buffer containing 32P-radiolabled cDNA probe (>109 cpm/µg DNA) at 68 C for 1 h. Membranes were subsequently washed three times in 2x saline-sodium citrate (SSC), 0.05% SDS, twice in 0.2x SSC, 0.1% SDS at room temperature for 10 min and then exposed to a Phosphorimager system screen for analysis (Molecular Dynamics, Inc., Sunnyvale, CA).

Statistical analysis
Data are expressed as mean and SD. Statistics were performed by ANOVA for dose-response curves and by Student’s t test to compare treated with untreated samples. Statistical significance is defined as a P value < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Phenotypic analysis of periosteum-derived cells grown in the presence of CDMPs and OP-1
Examination of the periosteal cultures treated with and without OP-1/CDMPs by light microscopy revealed the presence of two distinct types of cells: fibroblastic, and cells forming rounded nodules that stain positive for Alcian blue (1% at pH 2.5), indicating the presence of proteoglycans (Fig. 1, A and C). Alkaline phosphatase activity was detected in some rounded nodules and in fibroblast-like cells (Fig. 1BGo). The results of the RT-PCR analysis of messenger RNAs (mRNAs) for extracellular matrix proteins are shown in Fig. 1CGo. Periosteal cells, released by collagenase digestion and grown in monolayers as described in Materials and Methods, constitutively express osteocalcin, osteopontin and collagen type I which are characteristic for the osteogenic phenotype. Moreover, these cells also showed mRNA transcripts of cartilage markers such as aggrecan and collagen type II. This expression pattern appears to be independent of growth factor treatment and the time-point of evaluation (days 0, 2, 5, or 7).



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  		Figure 1. Phenotypic characterization of bovine periosteum-derived cells. A, Alcian blue staining of periosteum-derived cells after 7 days of culture on chamber slides in the presence of CDMP-1 (100 ng/ml). Magnification, x10. B, Alkaline phosphatase staining of cells released from bovine periosteum after 7 days of culture in the presence of 100 ng/ml CDMP-1. Magnification, x10. C, RT-PCR analysis of osteogenic and chondrogenic markers in bovine periosteum-derived cells cultured in the presence of CDMP-1, CDMP-2, and OP-1. Total cellular RNA was prepared from periosteum-derived cells grown to subconfluence in serum containing medium (lane 1), and from cultures switched to serum-free condition and incubated with 100 ng/ml of the indicated growth factors for 2 days (lanes 2–5), 5 days (lanes 6–9), and 7 days (lanes 10–13). Lanes 14 and 15 were from bovine chondrocytes (Cho) and bovine synoviocytes (Syn) and lane 16 shows negative controls (Neg. Co) of periosteum-derived cells, where the cDNA synthesis step was omitted. Reaction products specific for aggrecan, collagen type II, osteocalcin, osteopontin, collagen type I, and ß-actin were visualized on Sybr-green stained agarose gels. Co, Control; C1, CDMP-1; C2, CDMP-2.

 
CDMPs and OP-1 differentially stimulate cell proliferation of periosteum-derived cells
To evaluate the effects of CDMP-1, CDMP-2, and OP-1 on periosteal cell proliferation, mitogenic assays were performed after a 24-h serum-starvation period. The data showed that all evaluated growth factors stimulated [3H]-thymidine incorporation in a dose-dependent manner reaching statistical significance at 100 ng/ml for CDMP-1 (P < 0.05), 300 ng/ml for CDMP-2 (P < 0.05) and 10 ng/ml OP-1 (P < 0.05; Fig. 2Go). At the maximal concentrations of 300 ng/ml, OP-1 was the most potent stimulator of cell proliferation, followed by CDMP-1 and CDMP-2 when compared with unstimulated controls. These results indicate that both CDMPs as well as OP-1 resulted in a mitogenic response of periosteum-derived cells, with CDMP-2 being the least potent.



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  		Figure 2. Concentration-dependent stimulation of DNA synthesis and total cellular protein by CDMP-1, CDMP-2, and OP-1 in cultured bovine periosteum-derived cells. Serum-starved cultures of subconfluent periosteum-derived cells were incubated with 10, 30, 100, and 300 ng of CDMP-1 (gray bars), CDMP-2 (white bars) or OP-1 (black bars), respectively, for 24 h. Cells were labeled with [3H]-thymidine for the final 6 h as described in Material and Methods. Relative levels of cellular DNA synthesis were expressed as a percentage of serum-starved control. Data points represent the mean ± SEM of twelve samples from three experiments. *, P < 0.05 vs. control; **, P < 0.01 vs. control.

 
Effects of CDMPs and OP-1 on the osteogenic differentiation of cultured periosteum-derived cells
The periosteum is considered to contain undifferentiated mesenchymal precursor cells, which can undergo an osteogenic differentiation process under appropriate conditions. To compare the osteogenic activities of CDMP-1 and CDMP-2 with that of OP-1, alkaline phosphatase activity and mRNA levels for osteocalcin and collagen type I were assessed in this study.

Alkaline phosphatase activity is an early marker of osteogenic differentiation that spontaneously appeared during culture of periosteum-derived cells (35). After 2 days in the presence of 100 ng/ml of the indicated growth factors, the cells had low levels of alkaline phosphatase activity with no significant differences between the treatment groups. However, when cells were cultured for 5 days, CDMP-1 and OP-1 significantly increased enzymatic activity as indicated in Fig. 3AGo (P < 0.01), which was more pronounced after 7 days. Interestingly, CDMP-2 at 100 ng/ml did not affect alkaline phosphatase activity.



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  		Figure 3. Effect of CDMP-1, CDMP-2, and OP-1 on alkaline phosphatase activity in bovine periosteum-derived cells. A, Periosteum-derived cells in 96-well tissue culture plates were grown under serum-free conditions in the presence of CDMP-1 (solid squares), CDMP-2 (open squares) and OP-1 (solid triangle) at 10, 30, 100, and 300 ng/ml, respectively, for 7 days. AP activity was determined in quadruplicate cultures as described in Material and Methods. Data are expressed as mean ± SD and are representative of four independent experiments. Enzymatic activity is calculated as nanomoles of p-nitrophenol liberated per minute and milligram of total cellular protein (nmol p-NP/min/mg protein). B, Subconfluent periosteal-derived cells were cultured for 0, 2, 4, and 7 days with serum-free medium with or without (solid circles) the addition of 100 ng/ml CDMP-1 (solid squares), CDMP-2 (open squares) and OP-1 (solid triangle). Time-dependent stimulation of AP activity is expressed as the mean ± SD of four wells from a representative experiment and normalized to total cellular protein. *, P < 0.05 vs. control; **, P < 0.01 vs. control.

 
At the highest evaluated concentration of 300 ng/ml, OP-1 was 1.8- and 2.8-fold more stimulatory than CDMP-1 and CDMP-2, respectively (both P < 0.01). When compared with unstimulated controls, OP-1 treated cells at 300 ng/ml increased alkaline phosphatase activity up to 6-fold after 7 days of culture (Fig. 3BGo). These data indicate that CDMP-1 and OP-1 can regulate early osteogenic differentiation of periosteum-derived cells, while CDMP-2 appears to have significantly less effect.

Northern blot analysis of mRNA expression of osteocalcin and collagen type I
Bovine periosteum-derived cells showed only barely detectable signals of osteocalcin mRNA by Northern blot analysis (Fig. 4AGo). However, treatment with 100 ng/ml OP-1 for 7 days caused an increase of osteocalcin mRNA levels when compared with band density of untreated control (P < 0.01). Transcripts for osteocalcin could also be detected when cells were cultivated in the presence of CDMPs, but the hybridization signal did not differ significantly from controls (Fig. 4BGo). OP-1 also stimulated the mRNA levels of collagen type I in periosteum-derived cells. CDMP-1 treatment resulted in a moderate increase of signal intensity when compared with unstimulated controls, again, the effects were not significant (Fig. 4BGo).



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  		Figure 4. Northern blot analysis of osteocalcin and collagen type I mRNA expression in bovine periosteum-derived cells. A, Ten micrograms of total RNA from periosteum-derived cells grown for 7 days under serum-free conditions without (track 1) or with the addition of 100 ng/ml OP-1 (track 2), 100 ng/ml CDMP-1 (track 3), and 100 ng/ml CDMP-2 (track 4), were subjected to Northern blot analysis as described in Materials and Methods. Membranes were hybridized with radiolabled RT-PCR products for osteocalcin and collagen type I. ß-Actin expression levels are shown to verify equal loading of mRNA. B, Densidometric analysis of osteocalcin and collagen type I mRNA levels, normalized to ß-actin signals in bovine periosteum derived cells after stimulation with CDMPs and OP-1 at 100 ng/ml for 7 days. Data are the mean corresponding to three different experiments and are given in % of unstimulated control. **, P < 0.01 vs. control.

 
Increased macromolecule biosynthesis in periosteum-derived cells is induced by CDMPs and OP-1
To investigate the metabolic effects of CDMP-1, CDMP-2, and OP-1 on chondrogenic differentiation of periosteum-derived cells, we measured 35S-sulfate incorporation into newly synthesized macromolecules. Cells cultured for 7 days in the presence of recombinant growth factors showed a significant stimulation of 35S-labeled macromolecule biosynthesis with CDMPs being less stimulatory than OP-1 (P < 0.01; Fig. 5Go); a marked increase of 35S-labeling was seen for OP-1 at 10 ng/ml (P < 0.05). Significant stimulatory effects of CDMPs were detected only at a dose of 100 ng/ml (P < 0.01; Fig. 5Go) and higher concentrations of growth factors in cultures did not show any increase in the biosynthetic activity.



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  		Figure 5. Effects of CDMP-1, CDMP-2, and OP-1 on 35S-sulfate incorporation in bovine periosteum-derived cells. A, Monolayer cultures of bovine periosteum-derived cells were maintained for 7 days with 10, 30, 100, and 300 ng/ml of CDMP-1, CDMP-2 or OP-1 in serum-free medium. Parallel cultures without the addition of growth factors served as unstimulated controls. At the end of the culture period cells were labeled with 35S-sulfate for 6 h as described in Materials and Methods. Radioactive incorporation into newly synthesized macromolecules is normalized to total cellular protein. Bars represent the mean ± SD of four samples from a representative experiment. *, P < 0.05 vs. control; **, P < 0.01 vs. control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The data presented in this report indicate that cells derived from the periosteum, prepared and grown under the conditions described, express markers of the osteogenic and the chondrogenic lineage, and respond to various growth factors of the BMP-superfamily, as evidenced by the ability of CDMP-1, CDMP-2, and OP-1 to stimulate mitotic activity and osteogenic differentiation in vitro. In addition, we found that CDMPs as well as OP-1 enhance the production of sulfated macromolecules, most probably proteoglycans. Therefore, CDMPs and OP-1, both growth factors that have been implicated in joint formation and bone development, may act as local regulators of cells within the periosteum during bone formation and bone remodeling.

It is well established that cells of the periosteum have the ability to differentiate into the osteogenic lineage during bone growth, whereas during fracture repair they can develop both an osteoblastic and a chondrogenic phenotype (2, 3, 4, 5). Moreover, in vivo studies confirmed the presence of mesenchymal progenitor cells in the cambium layer of the periosteum, the regulating factors important for their transformation into a lineage-specific phenotype remain unknown. Our recent in vitro studies have shown the ability of CDMPs/BMPs to enhance the differentiation process of osteogenic precursors (27). Successful in vitro osteogenesis did not require the presence of CDMPs/OP-1 because periosteum-derived cells grown as monolayers endogenously expressed known osteogenic markers. The data are in agreement with studies from Izumi et al. (11) who showed the expression of alkaline phosphatase and collagen type I mRNA in cultured bovine periosteum-derived cells, and we further extended these observations by RT-PCR analysis showing transcripts for osteocalcin and osteopontin. Because this culture system is probably a mixture of cells at different stages of chondrogenic and osteogenic maturation, we were unable to study the effects of CDMPs/OP-1 on the induction of the osteogenic phenotype from undifferentiated progenitors. However, CDMPs and OP-1 increased alkaline phosphatase activity and OP-1 the expression levels of osteocalcin mRNA, suggesting that these growth factors may play a role as local modulators of osteogenic differentiation.

Endochondral bone formation requires mesenchymal condensation that prefigures the future configuration of skeletal elements (36). We speculate that nodule formation appearing in monolayer cultures may resemble the process of precartilaginous condensation as it occurs in the case of fracture repair in the adult organism (37). In this regard, various studies indicated the involvement of members of the BMP-family in endochondral bone formation (38). Data from the current study support the hypothesis that CDMPs and OP-1 enhance cell proliferation, nodule formation (data not shown) and the synthesis of extracellular matrix. Our data are in agreement with studies of Mayer and co-workers (39), who investigated the mitogenic effects of various BMPs on DNA synthesis in periosteal cells. They showed that OP-1 stimulated cell proliferation that fits exactly to the results of our in vitro model, though we observed a further increase at higher concentration of the morphogen. Moreover, both studies indicate that members of the BMP-family are distinct growth factors with a restricted spectrum of biologic activities which might be due to the ligand specificity among the BMP receptors.

The activity of members of the BMP family is mediated by the serin/threonin kinase receptors BMP-IA and IB that dimerize with a common BMP-receptor II upon ligand binding. Recently, BMPR-IA expression was detected in the native periosteum, and during early fracture healing, expression of BMPR-IA and -IB was up-regulated in cells at the proliferating osteogenic layer of the periosteum (40). BMPR-II was found to be colocalized with BMPR-IA and BMPR-IB (19). CDMPs binds preferentially to BMPR-IB/BMPRII, whereas OP-1 very poorly binds to BMPR-IA, but with high affinity to both the activin receptor-like kinase-2 (ALK-2) and BMPR-IB (31, 41). It is therefore possible that cultured periosteum-derived cells represent a healthy tissue related model that may explain the differences concerning the biologic activity of the growth factors in our culture system. Moreover, we do not rule out that other members of the BMP-family such as BMP-2 and BMP-4, which are also expressed during the early stages of fracture healing (19), have similar effects on periosteum-derived cells.

Alcian blue staining indicates that most of the extracellular matrix is localized within the nodules with only weak staining intensity in adherent fibroblast-like cells. Because alcian blue binds to proteoglycans of the extracellular matrix, we measured 35S-sulfate incorporation into sulfated macromolecules to quantify the matrix synthesis within the nodules. CDMPs and OP-1 increased nodule formation and 35S-sulfate incorporation into newly synthesized macromolecules. This leads us to speculate that these growth factors may influence the early steps of endochondral bone formation. Furthermore, in a gene therapy approach using primary rabbit mesenchymal cells of periosteal origin, transfection with OP-1 led to a loss of adherence from the culture dish with further mineralization of the nodules. These cells were used successfully to repair critical size defects suggesting that periosteal cells are potential candidates to deliver OP-1 at local sites in tissue engineering applications (42, 43).

In contrast to other models using periosteum-derived cells where chondrogenic differentiation failed to appear in monolayer cultures, cells of bovine origin express a cartilage phenotype, independent of growth factor treatment and culture conditions. Because collagen type II is also expressed in precartilaginous cells, only the coexpression of collagen type II with aggrecan, or the specific determination of collagen type IIB would serve as a reliable marker of a mature chondrogenic phenotype (44). RT-PCR analysis showing the presence of collagen type IIB and aggrecan indicates that high-density culture conditions are not necessary to develop a chondrogenic phenotype in bovine periosteum-derived cells.

In conclusion, the results demonstrate that bovine periosteum-derived cells grown in monolayer cultures contain cells committed to the osteogenic as well as to the chondrogenic lineage. The increase of the osteogenic specific molecules, as well as the enhanced mitogenic and metabolic activity in response to CDMP-1, -2 and OP-1 suggest a possible involvement of these growth factors in bone growth and fracture repair.


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  		Table 1. Densidometric analysis of RT-PCR products normalized to ß-actin bands

 

   Acknowledgments
 
We are grateful to Dr. R. Ziesche and Mrs. E. Hofbauer for the use of the Phosphorimager, and to Mr. and Mrs. Hödl (Fleischerei Hödl, Vienna, Austria) for their kind support. The authors thank Mrs. J. Peeters and Mr. C.W. Steiner for excellent technical assistance and Dr. Chee Keng Ng for help with the preparation of the manuscript. Recombinant growth factors were kindly provided by Creative BioMolecules, Inc. (Hopkinton, MA) and Stryker Biotech (Hopkinton, MA).


   Footnotes
 
1 This study was supported by Grant No. P12651-med from the Austrian Science Foundation. Back

2 These authors contributed equally to the work. Back

Received October 24, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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