- Email: [email protected]
Production of cartilage collagens during metaphyseal bone healing in the mouse
,I,S i2x ? s
W
Production of Cartilage Collagens during Metaphysea[ Bone Healing in the Mouse IIRO EEROLA*, HANNELEUUSITALO*, HANNU AROt and EEROVUORIO* Skeletal Research Program, ':-Department of Medical Biochemistry and Molecular Biology,University of Turku and Deparment of Surgery,Turku University Central Hospital, Turku, Finland
Abstract Small defects of unfractured bone are believed to heal without a cartilaginous intermediate. We have determined the extent of cartilage production in an experimental model of metaphyseal bone repair involving defects in both cortical and cancellous bone, but no fracture. Northern analyses revealed the presence of mRNAs for type X and II collagens in the repair tissue. Immunohistology confirmed subperiosteal deposition of both collagen types adjacent to the defect. While the mRNAs for the two collagen types peaked by one week of defect healing, immunodetectable type X collagen was not observed until the second week. The data suggest that reactivity of periosteum and activation of chondrogenesis and subsequent endochondral ossification programs are involved in murine bone repair regardless of defect type. Key words: bone, bone healing, cartilage, collagen, mouse, mRNA.
Introduction Healing of fractured bones proceeds by formation of an external callus tissue which undergoes differentiation along the same developmental pathway of chondrogenesis, osteogenesis and bone remodelling as seen during skeletal formation (Sandberg et al., 1993). In an attempt to enhance bone healing, orthopedic surgeons have developed fixation techniques of fractures which reduce callus size to a minimum and result in direct osteonal healing without a cartilaginous intermediate (Aro and Chao, 1993). The repair mechanism of fractured bone is thus dependent on the rigidity of fixation across the fracture site, which also largely determines the size of the fracture callus (Sandberg et al., 1993). When movement between bone ends is minimized, the fracture callus remains small, and little or no chondrocytic differenMatrix Biology Vol. 17/1998, pp. 317-320 © 1998 by Gustav FischerVerlag
tiation is observed (Glimcher et al., 1980; Lane et al., 1982). The extent of chondrogenesis and chondrocyte hypertrophy during bone healing can be studied by determining mRNA and/or protein levels of type II and X collagen, respectively (Hiltunen et al., 1993; Sandberg et al., 1993). Although the exact role of type X collagen in the endochondral ossification remains unknown, it is widely considered a specific marker for the process. Recently, the pattern of type X collagen expression has been extended to include articular cartilage which does not undergo endochondral ossification (Eerola et al., 1998). To further extend our knowledge of type X collagen and chondrocyte hypertrophy in adult cartilages, we employed Northern analysis and immunohistochemistry to study type X collagen expression in an experimental bone healing model in the mouse.
318
I. Eerola, H. Uusitalo, H. Aro and E. Vuorio
Materiats and Methods Experimental model for metaphyseal bone repair We used 32 C57 black/DBA mice, aged 3 mo . A round defect, 0.9 mm in diameter, was drilled under general anesthesia through the anterolateral cortical bone into the cancellous bone of the distal metaphysis of the femur above the growth plate; the limb cast. was immobilized for three weeks. The mice were killed 7, 14, 21 or 42 days after surgery and the defect areas dissected with 2 mm margins for RNA extraction and immunohistochemical examination.
Northern hybridizations The metaphyseal bone samples were randomly combined in pairs and powderized under liquid nitrogen for extraction of total RNA (Chirgwin et al., 1979). For Northern analyses, 10 ~g aliquots of total RNA were denatured by formaldehyde, electroforesed on 1% agarose gels and transferred to Pall Biodyne membranes (Pall Europe, Portsmouth, UK). Hybridizations and washes were performed as suggested by the supplier. 32p_ labeled inserts from cDNA clones pMColl0al-1 (Elima et al., 1992) and pMCol2al-1 (MetsS.ranta et al., 1991) were used as probes for type X and II collagen mRNAs, respectively. The bound probes were quantified on a Molecular Imager phosphoimager and mRNA levels calculated per 28S rRNA.
nous intermediate. In the present study, we observed transcriptional activation of type II and X collagen genes during repair of metaphyseal bone defects at postoperative days 7, 14, 21 and 42 (Fig. 1). In Northern analysis, type II and X collagen mRNA levels, which were essentially undetectable in normal metaphyseal bone, increased to their maximum level at day 7 of healing. Thereaftm, type II collagen mRNA levels gradually declined to a background level by postoperative day 42. Type X collagen mRNA levels remained at an elevated and relatively constant level until day 21, but they also dropped to background levels by day 42. The unexpected detection of type II and X collagen mRNAs in healing metaphyseal bone defects prompted us to perform immunolocalization of these collagen types. On the seventh postoperative dab both the medullary cavity and the cortical defect were filled with undifferentiated mesenchymal cells. In a few places, positive immunostaining for type II collagen and chondrocytic differentiation was observed subperiosteally adjacent to the cortical bone defect, but no staining for type X collagen was observed (Fig. 2A,B). At 14 days, new cancellous bone was seen in the medullary cavity, on the surface of the original cortex, and across the cortical defect. Cartilage staining positively for type II collagen ex-
A
7d
Type X ~
Immunohistochemistry For immunohistochemical stainings, the tissue samples were fixed in 4% paraformaldehyde, decalcified in 10% EDTA, embedded in paraffin and sectioned transversely. Monoclonal antibodies were used to detect type X (Girkontaite et al., 1996) and type II collagen (Linsenmayer et al., 1980) as described by Eerola et al. (1998).
The present study focuses on two aspects of bone defect repair: the involvement of endochondral ossification, and the temporal and spatial separation of chondrogenesis and cartilage hypertrophy during repair. While the role of endochondral ossification in skeletal growth and fracture repair is well established (Sandberg et al., 1993), healing of small defects of unfractured bone has been considered to proceed without a cartilagi-
14d
1
21d
..... ~
:
Type X collagen
C
[
42d
I
:
Type II 28S B
Type II collagen I
i o.
0.8 0.6 0.4 0.2 0
o. tbL
0.4 ~ 0.2
0
Results and Discussion
I
7
14
21
Postoperative lime (days)
42
0
7
14
21
42
Postoperative time (days)
Figure 1. Northern analysis of healing bone defects for type X and II collagen mRNA levels (A). Total RNA was isolated from three separate duplicate defect samples at 7, 14, 21 and 42 days of healing (shown above the lanes) and from four samples of unfractured bone (co). After electrophoresis and transfer to Pall Biodyne membrane, the samples were hybridized with ;:l~labeled probes for o~I(X) and prooN (II) collagen mRNAs, and for 28S rRNA. The hybridization signals (B and C) were quantiffed using a phosphoimager and the signals corrected for variations in the 28S rRNA levels.
Type X Collagen in Bone Repair
319
Figure 2. Immunohistological localization of type X and II collagens in healing metaphyseal bone defects at one (A and B), two (C and D) and three (E and F) weeks of defect repair. Serial sections were immunostained for type X collagen (A, C and E) and for type II collagen (B, D and F); p, periosteum; b, original cortical bone. Paraffin sections; immunostaining with avidin-biotin-peroxidase complex, counterstaining with hematoxylin; bar = 250 mm.
tended subperiosteally over the cortical defect (Fig. 2D). Type X collagen immunostaining was restricted to the innermost layers of this cartilage, populated by hypertrophic chondrocytes (Fig. 2C). By postoperative day 21, the area occupied by cartilage had increased to its maximum. The area staining positively for type X collagen overlapped that for type II collagen, but it was confined to areas around hypertrophic chondrocytes (Fig. 2E, 2F). No sign of cartilage or activation of the chondrogenetic program was seen within the marrow cavity. The data thus confirm that chondrogenic activation i.e., occurrence of cells exhibiting a chondrocytic pheno-
type surrounded by a matrix rich in type II collagen, which undergo hypertrophy and secrete type X collagen into the surrounding matrix (Fig. 2) - also takes place during healing of unfractured bone defects. In all cases analyzed, chondrocytic differentiation first occurred subperiosteally on the outer surface of the cortical defect, suggestive of periosteal origin of chondrogenic progenitors. During skeletal growth, cells located in the gambium layer of periosteum have been observed to contain type II and IX collagen mRNAs, which has been interpreted to indicate their prechondrogenic origin (Sandberg et al., 1993). The chondrogenic reactivity of perios-
320
I. Eerola, H. Uusitalo, H. Aro and E. Vuorio
teum has also been demonstrated by injection of saline or growth factors of the TGF-]3 family subperiosteally, which triggers chondrogenesis (Joyce et al., 1990). We feel our present observations are another indication of the potential of periosteum to feed chondrocyte precursors into repair tissue. Why cartilage production has not been observed in larger defects in chick, rat and rabbit (Glimcher et al., 1980; Lane et al., 1982), remains to be explained. This could be a feature specific for the murine species or a result of the more sensitive methods and systematic screening through the entire defect area employed in the present study. Another surprising finding was the rapid increase in type X collagen m R N A levels in healing metaphyseal bone defects: the maximal level was seen at day seven of healing although no type X collagen deposits could be identified at this point. While accumulation of specific mRNAs in cells prior to their translation and secretion has been observed during developmental processes, this has not been shown before for type X collagen. It remains to be shown whether the activation of the endochondral ossification program observed results from the model used, involving both cortical and cancellous bone, or from the species, the mouse, which has not been analyzed in this context before. The results also suggest that the critical size of the bone defect undergoing direct osteonal healing in the mouse is below 0.9 mm in diameter, i.e., much smaller than in other experimental animals (Glimcher et al., 1980; Lane et al., 1982).
Acknowledgements "]-he expert technical help of P/iivi Auho, Merja Lakkisto and Tuula Oivanen is gratefully acknowledged. This study was supported by the Academy of Finland, Sigrid Juselius Foundation and TULES graduate school (IE and HU). We are grateful to Dr. Klaus vonder Mark and Dr. Mikko l,ammi for the the antibodies.
References Aro, H.T. and Chao, E.Y.S.: Bone-healing patterns affected by loading, fracture fragment stability, fracture type, and fracture site compression. Clin. Orthop. 293: 8-17, 1990.
Chirgwin, I.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J.: Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5294-5299, 1979. Eerola, I., Salminen, H., Lammi, P., Lammi, M., yon der Mark, K., Vuorio, E. and S/i/im~inen, A.M.: Type X collagen is a natural component of mouse articular cartilage; association with growth, aging and osteoarthritis. Arthritis Rheum., in press, 1998. Elima, K., Mets/iranta, M., Kallio, J., Per/il/i, M., Eerola, 1., Garofalo, S., de Crombrugghe, B. and Vuorio, E.: Specific hybridization probes for mouse 0~2(IX) and oil(X) collagen mRNAs. Biochim. Biophys. Acta 1130: 78-80, 1992. Girkontaite, I., Frischholz, S., Lammi, P., Wagner, K., Swoboda, B., Aigner, T. and yon der Mark, K.: Immunolocalization of type X collagen in normal fetal and adult osteoarthritic cartilage with monoclonal antibodies. Matrix Biol. 15: 231-238, 1996. Glimcher, M., Shapiro, E, Ellis, R. and Eyre, D.: Changes in tissue morphology and collagen composition during the repair of cortical bone in the adult chicken..1. Bone Joint Surg. 62A- 964-973, 1980. Hiltunen, A., Aro, H. and Vuorio, E.: Regulation of extracellular matrix genes during fracture healing in mice. Clin. Orthop. 297: 23-27, 1993. Joyce, M.E., Roberts, A.B., Sporn, M.B. and Bolander, M.E.: Transforming growth factor-lB and the initiation of chondrogenesis and osteogenesis in the rat femur. J. Cell Biol. 110: 2195-2207, 1990. Lane, J.M., Golembiewski, G., Boskey, A.L. and Posner, A.S.: Comparative biochemical studies of the callus matrix in immobilized and non-immobilized fractures. Metab. Bone Dis. Rel. Res. 4: 61-68, 1982. Linsenmayer, T.,E and Hen&ix, M.J.: Monoclonal antibodies to connective tissue macromolecules: Type II collagen. Biochem. Biophys. Res. Commun. 92: 440-446, 1980. Mets/iranta, M., Toman, D., de Crombrugghe, B. and Vuorio, E.: Specific hybridization probes for mouse types I, II, 111 and IX collagen mRNAs. Biochim. Biophys. Acta 1089: 241-243, 1991. Sandberg, M., Aro, H.T. and Vuorio, E.: Gene expression during during bone repair. Clin. Orthop. 289:292-312, 1993.
Dr. Iiro Eerola, Department of Medical Biochemistry and Molecular Biology, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland.
Received February 12, 1998; accepted May 12, 1998.
W
Production of Cartilage Collagens during Metaphysea[ Bone Healing in the Mouse IIRO EEROLA*, HANNELEUUSITALO*, HANNU AROt and EEROVUORIO* Skeletal Research Program, ':-Department of Medical Biochemistry and Molecular Biology,University of Turku and Deparment of Surgery,Turku University Central Hospital, Turku, Finland
Abstract Small defects of unfractured bone are believed to heal without a cartilaginous intermediate. We have determined the extent of cartilage production in an experimental model of metaphyseal bone repair involving defects in both cortical and cancellous bone, but no fracture. Northern analyses revealed the presence of mRNAs for type X and II collagens in the repair tissue. Immunohistology confirmed subperiosteal deposition of both collagen types adjacent to the defect. While the mRNAs for the two collagen types peaked by one week of defect healing, immunodetectable type X collagen was not observed until the second week. The data suggest that reactivity of periosteum and activation of chondrogenesis and subsequent endochondral ossification programs are involved in murine bone repair regardless of defect type. Key words: bone, bone healing, cartilage, collagen, mouse, mRNA.
Introduction Healing of fractured bones proceeds by formation of an external callus tissue which undergoes differentiation along the same developmental pathway of chondrogenesis, osteogenesis and bone remodelling as seen during skeletal formation (Sandberg et al., 1993). In an attempt to enhance bone healing, orthopedic surgeons have developed fixation techniques of fractures which reduce callus size to a minimum and result in direct osteonal healing without a cartilaginous intermediate (Aro and Chao, 1993). The repair mechanism of fractured bone is thus dependent on the rigidity of fixation across the fracture site, which also largely determines the size of the fracture callus (Sandberg et al., 1993). When movement between bone ends is minimized, the fracture callus remains small, and little or no chondrocytic differenMatrix Biology Vol. 17/1998, pp. 317-320 © 1998 by Gustav FischerVerlag
tiation is observed (Glimcher et al., 1980; Lane et al., 1982). The extent of chondrogenesis and chondrocyte hypertrophy during bone healing can be studied by determining mRNA and/or protein levels of type II and X collagen, respectively (Hiltunen et al., 1993; Sandberg et al., 1993). Although the exact role of type X collagen in the endochondral ossification remains unknown, it is widely considered a specific marker for the process. Recently, the pattern of type X collagen expression has been extended to include articular cartilage which does not undergo endochondral ossification (Eerola et al., 1998). To further extend our knowledge of type X collagen and chondrocyte hypertrophy in adult cartilages, we employed Northern analysis and immunohistochemistry to study type X collagen expression in an experimental bone healing model in the mouse.
318
I. Eerola, H. Uusitalo, H. Aro and E. Vuorio
Materiats and Methods Experimental model for metaphyseal bone repair We used 32 C57 black/DBA mice, aged 3 mo . A round defect, 0.9 mm in diameter, was drilled under general anesthesia through the anterolateral cortical bone into the cancellous bone of the distal metaphysis of the femur above the growth plate; the limb cast. was immobilized for three weeks. The mice were killed 7, 14, 21 or 42 days after surgery and the defect areas dissected with 2 mm margins for RNA extraction and immunohistochemical examination.
Northern hybridizations The metaphyseal bone samples were randomly combined in pairs and powderized under liquid nitrogen for extraction of total RNA (Chirgwin et al., 1979). For Northern analyses, 10 ~g aliquots of total RNA were denatured by formaldehyde, electroforesed on 1% agarose gels and transferred to Pall Biodyne membranes (Pall Europe, Portsmouth, UK). Hybridizations and washes were performed as suggested by the supplier. 32p_ labeled inserts from cDNA clones pMColl0al-1 (Elima et al., 1992) and pMCol2al-1 (MetsS.ranta et al., 1991) were used as probes for type X and II collagen mRNAs, respectively. The bound probes were quantified on a Molecular Imager phosphoimager and mRNA levels calculated per 28S rRNA.
nous intermediate. In the present study, we observed transcriptional activation of type II and X collagen genes during repair of metaphyseal bone defects at postoperative days 7, 14, 21 and 42 (Fig. 1). In Northern analysis, type II and X collagen mRNA levels, which were essentially undetectable in normal metaphyseal bone, increased to their maximum level at day 7 of healing. Thereaftm, type II collagen mRNA levels gradually declined to a background level by postoperative day 42. Type X collagen mRNA levels remained at an elevated and relatively constant level until day 21, but they also dropped to background levels by day 42. The unexpected detection of type II and X collagen mRNAs in healing metaphyseal bone defects prompted us to perform immunolocalization of these collagen types. On the seventh postoperative dab both the medullary cavity and the cortical defect were filled with undifferentiated mesenchymal cells. In a few places, positive immunostaining for type II collagen and chondrocytic differentiation was observed subperiosteally adjacent to the cortical bone defect, but no staining for type X collagen was observed (Fig. 2A,B). At 14 days, new cancellous bone was seen in the medullary cavity, on the surface of the original cortex, and across the cortical defect. Cartilage staining positively for type II collagen ex-
A
7d
Type X ~
Immunohistochemistry For immunohistochemical stainings, the tissue samples were fixed in 4% paraformaldehyde, decalcified in 10% EDTA, embedded in paraffin and sectioned transversely. Monoclonal antibodies were used to detect type X (Girkontaite et al., 1996) and type II collagen (Linsenmayer et al., 1980) as described by Eerola et al. (1998).
The present study focuses on two aspects of bone defect repair: the involvement of endochondral ossification, and the temporal and spatial separation of chondrogenesis and cartilage hypertrophy during repair. While the role of endochondral ossification in skeletal growth and fracture repair is well established (Sandberg et al., 1993), healing of small defects of unfractured bone has been considered to proceed without a cartilagi-
14d
1
21d
..... ~
:
Type X collagen
C
[
42d
I
:
Type II 28S B
Type II collagen I
i o.
0.8 0.6 0.4 0.2 0
o. tbL
0.4 ~ 0.2
0
Results and Discussion
I
7
14
21
Postoperative lime (days)
42
0
7
14
21
42
Postoperative time (days)
Figure 1. Northern analysis of healing bone defects for type X and II collagen mRNA levels (A). Total RNA was isolated from three separate duplicate defect samples at 7, 14, 21 and 42 days of healing (shown above the lanes) and from four samples of unfractured bone (co). After electrophoresis and transfer to Pall Biodyne membrane, the samples were hybridized with ;:l~labeled probes for o~I(X) and prooN (II) collagen mRNAs, and for 28S rRNA. The hybridization signals (B and C) were quantiffed using a phosphoimager and the signals corrected for variations in the 28S rRNA levels.
Type X Collagen in Bone Repair
319
Figure 2. Immunohistological localization of type X and II collagens in healing metaphyseal bone defects at one (A and B), two (C and D) and three (E and F) weeks of defect repair. Serial sections were immunostained for type X collagen (A, C and E) and for type II collagen (B, D and F); p, periosteum; b, original cortical bone. Paraffin sections; immunostaining with avidin-biotin-peroxidase complex, counterstaining with hematoxylin; bar = 250 mm.
tended subperiosteally over the cortical defect (Fig. 2D). Type X collagen immunostaining was restricted to the innermost layers of this cartilage, populated by hypertrophic chondrocytes (Fig. 2C). By postoperative day 21, the area occupied by cartilage had increased to its maximum. The area staining positively for type X collagen overlapped that for type II collagen, but it was confined to areas around hypertrophic chondrocytes (Fig. 2E, 2F). No sign of cartilage or activation of the chondrogenetic program was seen within the marrow cavity. The data thus confirm that chondrogenic activation i.e., occurrence of cells exhibiting a chondrocytic pheno-
type surrounded by a matrix rich in type II collagen, which undergo hypertrophy and secrete type X collagen into the surrounding matrix (Fig. 2) - also takes place during healing of unfractured bone defects. In all cases analyzed, chondrocytic differentiation first occurred subperiosteally on the outer surface of the cortical defect, suggestive of periosteal origin of chondrogenic progenitors. During skeletal growth, cells located in the gambium layer of periosteum have been observed to contain type II and IX collagen mRNAs, which has been interpreted to indicate their prechondrogenic origin (Sandberg et al., 1993). The chondrogenic reactivity of perios-
320
I. Eerola, H. Uusitalo, H. Aro and E. Vuorio
teum has also been demonstrated by injection of saline or growth factors of the TGF-]3 family subperiosteally, which triggers chondrogenesis (Joyce et al., 1990). We feel our present observations are another indication of the potential of periosteum to feed chondrocyte precursors into repair tissue. Why cartilage production has not been observed in larger defects in chick, rat and rabbit (Glimcher et al., 1980; Lane et al., 1982), remains to be explained. This could be a feature specific for the murine species or a result of the more sensitive methods and systematic screening through the entire defect area employed in the present study. Another surprising finding was the rapid increase in type X collagen m R N A levels in healing metaphyseal bone defects: the maximal level was seen at day seven of healing although no type X collagen deposits could be identified at this point. While accumulation of specific mRNAs in cells prior to their translation and secretion has been observed during developmental processes, this has not been shown before for type X collagen. It remains to be shown whether the activation of the endochondral ossification program observed results from the model used, involving both cortical and cancellous bone, or from the species, the mouse, which has not been analyzed in this context before. The results also suggest that the critical size of the bone defect undergoing direct osteonal healing in the mouse is below 0.9 mm in diameter, i.e., much smaller than in other experimental animals (Glimcher et al., 1980; Lane et al., 1982).
Acknowledgements "]-he expert technical help of P/iivi Auho, Merja Lakkisto and Tuula Oivanen is gratefully acknowledged. This study was supported by the Academy of Finland, Sigrid Juselius Foundation and TULES graduate school (IE and HU). We are grateful to Dr. Klaus vonder Mark and Dr. Mikko l,ammi for the the antibodies.
References Aro, H.T. and Chao, E.Y.S.: Bone-healing patterns affected by loading, fracture fragment stability, fracture type, and fracture site compression. Clin. Orthop. 293: 8-17, 1990.
Chirgwin, I.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J.: Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5294-5299, 1979. Eerola, I., Salminen, H., Lammi, P., Lammi, M., yon der Mark, K., Vuorio, E. and S/i/im~inen, A.M.: Type X collagen is a natural component of mouse articular cartilage; association with growth, aging and osteoarthritis. Arthritis Rheum., in press, 1998. Elima, K., Mets/iranta, M., Kallio, J., Per/il/i, M., Eerola, 1., Garofalo, S., de Crombrugghe, B. and Vuorio, E.: Specific hybridization probes for mouse 0~2(IX) and oil(X) collagen mRNAs. Biochim. Biophys. Acta 1130: 78-80, 1992. Girkontaite, I., Frischholz, S., Lammi, P., Wagner, K., Swoboda, B., Aigner, T. and yon der Mark, K.: Immunolocalization of type X collagen in normal fetal and adult osteoarthritic cartilage with monoclonal antibodies. Matrix Biol. 15: 231-238, 1996. Glimcher, M., Shapiro, E, Ellis, R. and Eyre, D.: Changes in tissue morphology and collagen composition during the repair of cortical bone in the adult chicken..1. Bone Joint Surg. 62A- 964-973, 1980. Hiltunen, A., Aro, H. and Vuorio, E.: Regulation of extracellular matrix genes during fracture healing in mice. Clin. Orthop. 297: 23-27, 1993. Joyce, M.E., Roberts, A.B., Sporn, M.B. and Bolander, M.E.: Transforming growth factor-lB and the initiation of chondrogenesis and osteogenesis in the rat femur. J. Cell Biol. 110: 2195-2207, 1990. Lane, J.M., Golembiewski, G., Boskey, A.L. and Posner, A.S.: Comparative biochemical studies of the callus matrix in immobilized and non-immobilized fractures. Metab. Bone Dis. Rel. Res. 4: 61-68, 1982. Linsenmayer, T.,E and Hen&ix, M.J.: Monoclonal antibodies to connective tissue macromolecules: Type II collagen. Biochem. Biophys. Res. Commun. 92: 440-446, 1980. Mets/iranta, M., Toman, D., de Crombrugghe, B. and Vuorio, E.: Specific hybridization probes for mouse types I, II, 111 and IX collagen mRNAs. Biochim. Biophys. Acta 1089: 241-243, 1991. Sandberg, M., Aro, H.T. and Vuorio, E.: Gene expression during during bone repair. Clin. Orthop. 289:292-312, 1993.
Dr. Iiro Eerola, Department of Medical Biochemistry and Molecular Biology, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland.
Received February 12, 1998; accepted May 12, 1998.