Type X collagen synthesis during endochondral ossification in fracture repair.

Collagen synthesis in normal connective tissue development and repair is integral to tissue stability. The appearance of a short chain collagen, designated Type X, was studied in experimental fractures created in the chicken humerus. Biosynthetic studies using [14C]proline incorporation coupled with histologic examination of the cartilaginous callus demonstrated that Type X collagen synthesis occurs during endochondral ossification in the fracture callus. Type X synthesis occurred in the areas of cartilaginous callus composed of hypertrophic and degenerative chondrocytes that were associated with increased vascularity and matrix mineralization. Synthesis of short chain collagen was not detected in either skeletal muscle or bone. Two-dimensional peptide mapping of cyanogen bromide and proteolytic fragments derived from fracture callus short chain collagen confirmed the identity of this collagen as Type X. The synthesis of Type X collagen by fracture callus is further evidence supporting its close association with the process of endochondral ossification.

Collagen synthesis in normal connective tissue development and repair is integral to tissue stability. The appearance of a short chain collagen, designated Type X, was studied in experimental fractures created in the chicken humerus. Biosynthetic studies using [14C]proline incorporation coupled with histologic examination of the cartilaginous callus demonstrated that Type X collagen synthesis occurs during endochondral ossification in the fracture callus. Type X synthesis occurred in the areas of cartilaginous callus composed of hypertrophic and degenerative chondrocytes that were associated with increased vascularity and matrix mineralization. Synthesis of short chain collagen was not detected in either skeletal muscle or bone.
Two-dimensional peptide mapping of cyanogen bromide and proteolytic fragments derived from fracture callus short chain collagen confirmed the identity of this collagen as Type X. The synthesis of Type X collagen by fracture callus is further evidence supporting its close association with the process of endochondral ossification.
Several extracellular matrix macromolecules are synthesized at various developmental stages in normal chondrogenesis, limb growth, and maturation of the skeleton (1). Within cartilage, collagens comprise approximately 40-50% of its dry weight, the predominant form being Type I1 collagen. Several other collagens are present in cartilage in smaller amounts than Type 11, these include la,2a,3a, high molecular weight and low molecular weight collagen, classified as Type IX collagen and a short chain collagen, referred to as Type X (2).
Alterations in the appearance of some of these collagens (3)(4)(5)(6)(7)(8) occur during cell differentiation and may be involved with changes in cell phenotype (9). Type X collagen is synthesized in the cartilaginous tissues of the growth plate (3,10,11) and the calcifying region of the sternum (12) and appears to be unique to the process of endochondral ossification.
Endochondral ossification is not restricted to the growth plate, but also occurs during the process of fracture healing with transition of a cartilaginous callus intermediate to bone (13)(14)(15). In this study, a fracture callus model was used to investigate Type X collagen synthesis. Our data demonstrate the synthesis of Type X in fracture callus primarily within *This work was supported by Grants T32-AM07482 and R01-AM29766 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisenent" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 9 To whom correspondence and reprint requests should be addressed Orthopaedic Research Laboratory, University of Virginia School of Medicine, Box 374, Charlottesville, VA 22908. regions of the callus composed of hypertrophic cells and matrix that is undergoing vascularization and mineralization.

MATERIALS AND METHODS
Animal Model-Closed midshaft humerus fractures created in skeletally mature White Leghorn chickens were nonrigidly immobilized with orthopaedic stockinette about the thorax and the involved wing. Radiographs were obtained just prior to fracture, immediately postfracture, and at death 10 days later (see Fig. 1) at which time the fracture callus was freshly dissected from the bone fragments. The callus was divided into three tissue subgroups according to color, consistency, and vascularity using the dissecting microscope (13). Skeletal muscle and cortical bone were obtained from the contralateral limb as controls.
Organ Cultures and Extractiom-In all studies, representative portions of fracture callus were embedded in paraffin, sectioned, and stained with hematoxylin and eosin for histologic examination. The remainder of the tissue was placed in Dulbecco's modified Eagle's medium containing 25 mM HEPES,' and supplemented with ascorbic acid (50 pglml), P-aminoproprionitrile (50 pg/ml), penicillin (10,000 units/ml), streptomycin (10,000 pg/ml), and then preincubated at 37 "C for 1 h. The medium was removed and replaced with the same tissue culture medium supplemented with 5 pCi/ml of [U-'4C]proline (New England Nuclear). The tissue was incubated for 20 h in this medium and rinsed with 0.15 M NaCl, 0.05 M Tris(hydroxy-methy1)aminomethane-HC1 pH 7.5 (TBS) at 4 "C.
Subsequent to metabolic labeling, the fracture callus samples were individually homogenized in 1.0 M NaCl, 0.05 M Tris, pH 7.5, supplemented with protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 mM N-ethylmaleimide, and 25 mM EDTA) using a Tissunizer (Tekmar Co.) at 4 "C, stirred for 1 h, and centrifuged at 30,000 X g for 30 min. Supernatants were dialyzed against TBS containing 0.2 mM phenylmethylsulfonyl fluoride at 4 'C, and the proteins were precipitated by the addition of ammonium sulfate (33% final saturation) (3).
Limited pepsin proteolysis was conducted following dialysis of samples against 0.5 M acetic acid, pH 2.5. Digestion of extracted sample with 100 pg/ml pepsin (Worthington) was performed for 18 h at 4 "C, and the reaction was terminated by dialysis against TBS. Digestion with bacterial collagenase (Advanced Biofactures, Form 111) was carried out using both non-pepsin-and pepsin-treated fracture callus collagens. Forty to sixty units of collagenase in 20 p1 of 10 mM calcium acetate, 25 mM Tris, pH 7.4, was added to 10,000 dpm of substrate in 400 p1 of TBS, 10 mM N-ethylmaleimide, 0.2 mM phenylmethylsulfonyl fluoride and incubated at 37 "C for 4 h, and the reaction was stopped by freezing at -20 "C. Enzymatically digested and control samples were precipitated by the addition of trichloroacetic acid to a 10% final concentration.
Chondrocyte Cultures-Chondrocytes were prepared from the region of presumptive calcification of 17-day-old chick embryo sterna (12) and grown in monolayer culture for 2 weeks. Sternae were incubated with 1500 units of hyaluronidase (HSE, Cooper Biomedical) for 20 min, rinsed, and incubated for 90 min with a mixture of one part of 4000 units of bacterial collagenase (CLSPA, Cooper Biomedical) to two parts 0.5% trypsin (GIBCO). The cells were centrifuged for 10 min at approximately 300 X g, rinsed with saline, and seeded at a cell density of 7 X IO4 per cm2 in Dulhecco's modified Eagle's medium containing 25 mM HEPES supplemented with 10% fetal hovine serum. To prepare radioactively labeled collagens, cultured cells were incuhated with 5 pCi/ml of ["Clproline for 24 h, the culture medium was collected into protease inhihitors, and the proteins were precipitated hv the addition of 10% trichloroacetic acid. ( k / I.:/ec~rophoresis-Organ culture and cell culture medium proteins and the products from enzymatic digestion were analyzed hy SDS-PAGE on 7.S"; slab gels (16). Gels were stained with Coomassie Hrilliant Blue R-250, destained in 10% methanol, i % acetic acid, and proressed with EN'IHANCE (New England Nuclear) for fluorography. Presensitized S-Omat AR x-ray film (Kodak) was exposed at -70 "C (17).
Quantitative estimation of radioactivity within individual hands was performed hy densitometric scanning (E-C Apparatus Corp.) and electronic integration (SP-4270, Spectra-Physics). Fracture callus collagens and collagens prepared from culture medium of calcifying Iy-day chick emhryo sternal chondrocytes were analyzed hy twodimensional SDS-PAGE using 50,000 dpm/lane of pepsin-treated material. Electrophoresis in the first dimension was performed on i . 5 7 SDS-PAGE in the presence of 50 mM dithiothreitol. After removing these lanes from the gel, each lane was washed three times with 70'~; formic acid and then placed in a solution of cyanogen hromide at 10 mg/ml in 70% formic acid for 2 h a t room temperature while shaking. Following digestion, the lanes were rinsed with water and equilihrated with SDS-PAGE sample buffer containing 100 mM dithiothreitol. The lanes were then placed horizontally in contact with a separate second dimension 5% stacking, 12.5% separating gel and electrophoresed (18). A similar two-dimensional SDS-PAGE technique was used with Staphylococcus aureu.7 V-8 protease (500 pg/ ml) to compare peptide fragments from fracture callus collagens to those synthesized hv calcifying chick sternal chondrocytes.
Molecular weights of collagen chains were estimated using cyanogen hromide peptides of T-ype I collagen extracted from rat tail tendon and of T?ipe I1 collagen from rabbit structural cartilage. Peptides were prepared by digestion in 70% formic acid with an equal weight of cyanogen bromide for 4 h a t 30 "C.

RESULTS
Fracture Callus Collagen Synthesis Related to Histologic Regions-Representative histologic sections made of the three tissue subgroups dissected from 10-day chicken fracture callus are shown in Fig. 2. Tissue sample (subgroup) 1 was grossly white, avascular, and, upon microscopic examination, consisted of fibrous tissue and fibrocartilage with areas of residual skeletal muscle. Sample 2 was chondroid, more transparent, and had few areas of vascular invasion. Many of the chondroc-ytes were undergoing h-ypertrophv and a few areas of calcified matrix were seen. Sample 3 was more vascular. consisted primarily of hypert.rophic chondroc-des, and had large areas of calcified matrix and immature bone.
Metabolic labeling with [l"C]proline followed by SDS-PAGE demonstrated that T-ype I collagen was predominant in bone, muscle, and the first callus sample, whereas T-ype I1 collagen synthesis was increased in the hyaline cartilaginous areas of the callus represented by samples 2 and 3 (see Fig.  3). Synthesis of the 55-kDa form of a short chain collagen was initially observed in t,he second sample of the fracture callus and became the major form in association with h-ypertrophic chondrocytes and matrix mineralization in sample 3 (Fig. 3, lanes 2 and 3).
Densitometric scanning of several fluorograms was used to quantitate the relat.ive densities of a chain and short chain collagen bands for the three callus subgroups (see Table I).
The ratio of nl:n2 calculated for the first subgroup was 2.0, consistent with the synthesis of T-ype I collagen, the expected product of fibrous tissue and fibrocartilage. For subgroup 2, the ratio was 5.2, indicating a relative increase in the svnthesis of nl chains making up T-ype I1 collagen. This value decreases in subgroup 3 to 2.9, presumably due to svnt,hesis of the T.ype I collagen of osteoid. The short chain collagen comprised approximately 15% of the total radioactivity incorporated into the collagens of the second subgroup, and increased to 31% in the third subgroup.
Comparison of Fracture Callus Collagens with Collagen from Cultured Chondrocytes- Fig. 4 demonstrates the electrophoretic patterns of radiolabeled proteins synthesized by fracture callus in organ culture (lanes I and 2) compared with proteins synthesized and secreted into the medium of cukured sternal chondrocytes derived from the region of "presumptive calcification" of 17-day-old chick sterna (lanes 3 and 4). Lanes 2 and 4 represent the products of limited pepsin digestion of fracture callus and cell culture proteins, respectively. The major collagens synthesized by fracture callus include Type I, as noted by the presence of cul and n2 chains, and of Type I1 collagen, as indicated by the rat.io of c u l to cu2 being greater I.'I(., 2. l.'racture callus histology. Tetl-(I+> t111(is11;111 I I~I~I I ( ,~I I . I r w~u r e callus was sq):trate(i r l~~d c r t lw dissecting microscope into three subgroups 01: the hasis o f color, transparency, texture, and vascularity. Histologic study of three representative regions demonstrated: 1, lil~rous tissue and fihrocartilage; 2, prolilerating and hypertrophic chondrocytes with some areas of calcified matrix; and 9 , h.ypertrophic and degenerative chondrocytes with extensive areas of calcilied matrix. than 2:l. The non-disulfide-bonded short chain collagen was synthesized by this tissue with an estimated molecular mass o f 5.5 kDa (by collagenous standards) and was converted to 30 kDa by limited pepsin proteolysis. Digestion of the [''C] proline-labeled fracture callus collagens with bacterial collagenase demonstrated the sensitivity of both 5.5 kDa and 40 kDa in addition to T-ypes I and I1 (data not shown). The short chain collagen of chicken fracture callus migrates on SDS-PAGE with a 3-4-kDa smaller apparent molecular mass than the Type X collagen isolated from cell culture. Such small differences in molecular mass could reflect differences in posttranslational modification, such as hydroxylation or glycosylation. that may exist between the cell culture and organ culture systems.
C.vanogen Hromidc Iligest Peptide Mapping-To further establish the identity of this fracture callus short chain collagen as Type X, cyanogen bromide digestion peptides of t.he 40-kDa form (pepsinized) were compared to the peptides from a similar digestion of (pepsinized) embryonic chick sternal chondrocyte T-ype X collagen. The peptides resulting from cyanogen bromide digestion of fracture callus and sternal chondrocyte collagens were analyzed by two-dimensional gel electrophoresis (Fig. 5). The digestion products demonstrate at least eight definite peptide fragments. The correspondence between the cyanogen bromide peptide maps for these t.wo short chain collagens indicates that the fracture callus short chain collagen is a product of the Type X collagen gene.
Using the same two-dimensional SDS-PAGE technique, S. a u r w s V-8 protease was used to produce peptide fragments of the collagen chains. Peptide maps of the fracture callus short chain collagen and chondrocyte culture medium Type S were also similar using V-8 protease, confirming the results with cyanogen bromide cleavage (data not shown).

DISCUSSION
Type X collagen synthesis has been previously associated with endochondral bone formation in the developing chicken embryo tibiotarsus (10, ll), chondrocytes in culture derived from the "presumptive calcification" region of the sternum (12), and the h-ypertrophic region ofthe growth plate in several mammals (3,6,19). Our previous study further determined that synthesis of T-ye X collagen is limited within the growth plate to the zones of provisional calcification and "degenera-  (14), our histologic studies substantiate the role of chondroc-ytes in fracture callus to be similar to the cells in the hypertrophic region of the growth plate. The primary difference is the organization within fracture callus of numerous centers of ossification rather than the uniform transition to a calcified matrix that is seen in the growth plate. Similar histologic changes also occur during the induction of new bone by subcutaneously implanted demineralized bone matrix (20). This involves a transition process in which chondroblasts are first seen at day 5 following implantation, then proliferate, and by day 9 are associated with calcifying matrix.
Biochemical studies of fracture callus regions have shown parallels between this tissue and zones of the growth plate with respect to concentration of enzymatic and metabolic intermediates (14, 15). Analysis of specific collagen types in fracture callus demonstrates the synthesis of Type X collagen during matrix calcification, similar to processes in the growth plate. Type X collagen is not synthesized by bone, skeletal muscle, or by the fibrocartilaginous component of fracture callus. This collagen appears t.o be a consistent marker of endochondral ossification.
Matrix vesicles have been identified within the extracellular environment of chondroc-ytes (21,22). These membranous structures are thought to be derived from cLytoplasmic processes of chondrocyt.es and have been implicated with a role in t,he calcification of the vertical septa of growth plate There is a relative increase in T.ype 11 collagen svnthesis seen in samples 2 and 3 of fracture callus. Short chain collagen, svnthesized as a 55-kDa component, is initially seen in fracture callus sample 2 and to an even greater extent in sample 3 (consisting of hypertrophic chondrocvtes and large areas of calcified matrix). Following pepsin digestion to remove non-triple helical sequences. short chain collagen is converted to a 40-kDa component.

?'ABLE I f~rr~.sitornrtric yunntitntion offracturr cnlllls collagens
Several Ilnorngrams of fracture callus radiolaheled collagens were scanned with a densitometer and the relative percent densities of the sperilir chains and o f the short chain collagen bands were averaged. of Type X "procollagen" (59 kDa) in chick tibiotarsal explants of the h-ypertrophic region to a 49-kDa polvpeptide. and. more rerentlv, the same group has identified precursor components t o T?ipe X (25). Rernington et al. (19) reported the synthesis of a 180-kDa precursor to Type X collagen in rahhit growth plate. hut. did not observe processing beyond BO kDa. We have been unable to detect either a larger precursor form of the Type X collagen chain in bovine growth plate organ culture nor have we heen ahle to demonstrate processing of the initial form of the T-ype X collagen chain to a smaller species even after a 20-h chase in culture ( 3 ) . Likewise. in the 24-h fracture callus metabolic labelings, no evidence for proteolytic conversion could be detected bv analysis on SDS-PAGE.
Comparison of chick T-ype X collagen synthesized in cultures of chondrocytes from the "presumptive calcification" area of sterna to T-ype X synthesized in fracture callus revealed a 3-4-kDa difference between the two. the molecular mass of the fracture callus form being smaller. This mav be due to differences in post-translat.iona1 modification that may be inherent to unique characteristics of the two organs studied, the fracture callus wrsus the sternum. Alternativelv, we cannot rule out the possibilitv of differences between protein synthesis in organ culture compared to conditions in cell culture that mav contribute to changes in mobility. However, there were no obvious differences in the cyanogen bromide  (-) and after (+) digestion with pepsin either with (+) or without (-) the disulfide bond reducing agent dithiothreitol (DT7'). The non-disulfide-bonded 55-kDa short chain collagen synthesized by fracture callus is converted to 40 kDa following pepsin digestion. Type X collagen from culture medium of chick sternal chondrocytes has a slightly larger molecular mass, 58 kDa, than that seen in fracture callus. This is true also for the pepsin-digested form (X,,) with an apparent molecular mass of 44 kDa.

A B
FIG. 5. Two-dimensional mapping of cyanogen bromide peptides. Pepsin-digested collagens from fracture callus region 3 organ cultures ( A ) and culture medium of chick embryo sternal chondrocytes from the region of "presumptive calcification" ( R ) were run in the first dimension on SDS-PAGE using a 7.5% acrylamide gel, the lanes were excised and treated with cyanogen bromide, and the resultant peptide fragments were analyzed in the second dimension on a 12.5% acrylamide gel. The cyanogen bromide peptides from fracture callus short chain (40 kDa) and sternal chondrocyte Type X (Xwp) show extensive similarities. peptide maps that would explain the observed difference in molecular weight,.
Further knowledge of the properties of Type X collagen will help establish its role in endochondral ossification. Interactions between this collagen and other noncollagenous proteins (26-28), proteoglycans, or growth factors (29) could play a part in endochondral ossification. The association of T-ype X collagen synthesis in cartilage matrix mineralization may be a molecular marker that would distinguish between successful fracture repair and nonunion. Identification of environmental factors that induce synthesis of T-ype X collagen by fracture callus may he important in the treatment of fractures.
The formation of a cartilaginous callus during fracture healing may be favored by the mechanical movement of bone (30). A recent report indicates that Type I1 collagen detected by immunolocalization appears only in areas of motion during fracture repair (31), another describes changes in collagen composition during the repair of small defects in cortical hone without endochondral ossification ( 3 2 ) . In our present study, we used a nonrigidly immobilized fracture allowing a certain degree of motion. The appearance of Type X collagen synthesis in the areas showing hypertrophic chondroc-ytes and chondroid tissue with the concomitant svnthesis of Type I1 may be dependent on the restricted movement that was permitted to occur in our system. Utilization of a model in which hone repair can be studied a t a mechanically stable site could further elucidate the biomechanical conditions that stimulate the synthesis of specific collagen types and the ensuing cartilaginous tissue.