- Email: [email protected]
Induction of new bone by ceramic bovine bone with recombinant human bone morphogenetic protein 2 and transforming growth factor ß
Copyright 9
Int. J. Oral Maxillofac. Surg. 1998; 27." 310-314 Printed in Denmark. All rights reserved
lnternan'onalJournal of
Ord & Maxillo[acidSurgery ISSN 0901-5027
Induction of new bone by ceramic bovine bonewith recombinant human bone morphogenetic protein 2 and transforming growth factor/
Xlaohui Si, Yan Jin, Llanjla Yang Department of Oral Pathology, Stomatological College, Fourth Military Medical University, Xi'an, PR China
X. Si, Y. Jin, L. Yang: Induction of new bone by ceramic bovine bone with recombinant human bone morphogenetic protein 2 and transforming growth factor ft. Int. J. Oral Maxillofac. Surg. 1998; 27: 310-314. 9 Munksgaard, 1998 Abstract. Bone mt)rphogenetic protein (BMP) has the ability to induce ectopic bone, while the action of transforming growth factor fl (TGF t5) is to stimulate proliferation of osteoblasts and chondrocytes as well as the production of extracellular matrix. The aim of the present study was to study their synergistic actions in bone formation. Three kinds of complexes, recombinant human BMP2 (rhBMP2), T G F fl and rhBMP2frGF fl in ceramic bovine bone (CBB), were made and then implanted into the thigh muscle pouches of mice. The histological reactions of the implanted areas were studied at intervals of 3, 5, 7, 14 and 21 days. The results showed that, except for the implants with T G F fl alone, both rhBMP2 and rhBMP2/TGF fl implants exhibited new ectopic bone formation. The morphometric study revealed that the quantity of newly formed bone induced by rhBMP2/TGF fl was obviously greater than by rhBMP2 alone. These results indicate that T G F fl in combination with BMP may enhance formation of ectopic bone.
A variety of bone growth factors play a role in bone morphogenesis, development, growth, repair and remodelling. It is apparent that bone contains a number of structurally related proteins, including bone morphogenetic protein (BMP) and transforming growth factor fl (TGF fl), that are involved in bone formation 3. BMP and T G F fl belong to the T G F fl superfamily. This family is composed of at least 25 members of the molecules which retain the seven cysteine residues within the mature region of the protein 9,24. Of these, BMP and T G F fl are receiving considerable attention for potential clinical application. BMP is a protein which can induce new
cartilage and bone formation in extraskeletal tissues by stimulating undifferentiated mesenchymal cells to proliferate and differentiate into bone-forming cells. Human cDNA of BMP1 and eight other related BMPs (BMP2BMP9) have been cloned and expressed 4,23. Recombinant human BMP2 (rhBMP2) is considered to be the most active and could induce new bone formation at both bony and nonbony sites alone. T G F fl is present in most tissues and is known to play an important regulatory role in cell growth, migration and differentiation. Platelets are a major storage site for this growth factor, and they contain 10-100-fold more
Key words: bone morphogeneUc protein; transforming growth factor 13;bone induction; ceramic bovine bone. Accepted for publication 30 December 1997
T G F fl than do the other tissues 2. T G F fl has been shown to promote bone formation and to enhance bone healing, and is present in bone in high quantities 19. In vitro and in vivo studies have indicated that T G F ] / h a s stimulatory effects on proliferation and extracellular matrix synthesis in many types of skeletal tissue cells. BMP and T G F fl may be synergistic in promoting bone formation because of their effects on different stages of cells. The combination of BMP and T G F fl might result in better bone growth than each of them separately. Thus, the aim of the present study was to observe the BMPbone induction influenced by T G F ft.
New bone formation induced by r h B M P 2 and TGF fl Materials and methods Growth factors
TGF fl was obtained from fresh bovine blood platetets by acid/ethanol procedure and chromatography. The bioassay of TGF fl determined the ability of the polypeptide to induce anchorage-independent growth in nonneoplastic NRK fibroblasts by measuring the formation of colonies of cells in soft agar2. An in vivo study of TGF fl was performed as described earlierIs and it showed that TGF fl could cause the formation of granulation tissues at the site of injection in newborn mice. rhBMP2 is the purified recombinant protein from E. coli, and was kindly provided by Dr M. Zhao (Institute of Basic Medical Science, Academy of Military Medical Science, PR China). The in vivo ectopic bone formation assay revealed that this material was active at 0.5 rag, when tested seven days after implantation12.
Complex preparation
The blocks of multipore ceramic bovine bone (CBB) were produced from fresh bovine rib bones which consist of a cortical layer and a cancellous part. The resected ribs were prepared by boiling them and cleaning them from the periosteum and adjacent connective tissues and then cut into blocks. The blocks were washed in normal saline, soaked in H202 to remove proteins and then washed in running water. The deproteinized blocks were heated at 900~ for 1 h. Each block weighed about 20 mg and was selected as the carrier for combining rhBMP2 and/or T G F ft. Three kinds of complexes were made: rhBMP2 and CBB (rhBMP2/CBB), CBB and the purified bovine T G F fl ( T G F fl/CBB), and rhBMP2/TGF fl/
311
CBB. The CBB implants were coated with rhBMP2 and/or T G F fl under aseptic conditions by soaking them in solutions containing rhBMP2 and/or T G F fl for 18 hours at 4~ and then lyophilized. The dose of T G F fl and rhBMP2 for combination with each CBB block were 15 ng and 2 mg respectively. Animal procedures and histological examination
A total of 45 mice were randomly divided into three experimental groups for the implantation of rhBMP2/CBB (Group 1), TGF/CBB (Group 2), and rhBMP2/TGF/CBB (Group 3). The mice were anesthetized with ether and the above complexes were implanted into their right thigh muscle pouches. Each complex was implanted into 15
Fig. 1. rhBMP2/TGF fl/CBB implantation (day 3). ThiS section shows aggregation of mesenchymal cells in muscle (HEX400).
Fig. 2. rhBMP2/rGF fl/CBB implantation (day 5). Proliferation of mesenchymal cells is visible (HEX400).
Fig. 3. rhBMP2/TGF fl/CBB implantation (day 7). Mesenchymal cells differentiated into chondrocytes (HE•
Fig. 4. rhBMP2/TGF fl/CBB implantation (day 14). Newly formed bone is seen (HEx200).
312
Si et al.
Fig. 5. rhBMP2/CBB implantation (day 14). rhBMP2 alone exhibited
low levels of woven bone (HE•
animals. The animals were killed at intervals of 3, 5, 7, 14 and 21 days after implantation. Tissues were fixed for 24 h in 10% formalin, followed by decalcification in formic acid/formalin for 72 h. Paraffin-embedded specimens were stained with hematoxylin and eosin. The area of newly formed bone by day 14 was measured-by using the lattice in the eye-piece of the microscope (10x). The data were analysed by using Statgraph software. Results
There was no new bone formation in group 2, i.e. TGFfl/CBB blocks. Proliferation of fibroblasts was seen by day 3 and day 5 and a thin condensed layer of fibrous connective tissue, surrounding the implanted blocks, by day 21. Both group 1 and group 3 showed aggregation and proliferation of round and spindle-shape mesenchymal cells by day 3 and day 5 (Figs. 1, 2). There was mild inflammatory cell infiltration in the implanted areas. By comparison, the inflammatory reaction was less in group 3 than in group 1 five days postimplantation. The inflammatory reaction disappeared by day 7 and both showed formation of new cartilage (Fig. 3) and woven bone with newly formed marrow by day 14 (Figs. 4, 5). The newly formed bone trabeculac were thicker in ,group 3 than in group 1. The newly formed bone marrow of group 3 appeared a few days earlier than in group 1. By day 21, newly formed bone became mature and cortical bone with bone marrow was observed (Fig. 6). Newly
Fig. 6. rhBMP2/TGF fl/CBB implantation (day 21). Mature cortical bone has formed (HE•
formed bone tissue was detected throughout the full thickness of the implants of rhBMP2/CBB and rhBMP2/TGF fl/CBB. A delicate reticular network of newly formed bone was seen in the pores of the central area of the CBB. A morphometric study revealed that the average area of newly formed bone by day 14 in group 3 was 0.223 mm 2, whereas in group 1 it was 0.030 mm 2. Analysis showed the difference between group 3 and group 1 to be statistically significant (t-test, Table 1). Discussion
BMP irreversibly induces differentiation of perivascular mesenchymaltype cells into cartilage and bone-forming cells. It is capable of inducing ectopic bone formation in vivo. Low dosage of native allogeneic BMP showed strong osteoinductivity and low immunogenicity21. The sequence of cctopic bone formation induced by BMP is similar to that of embryonic bone formarion and fracture repair. The sequential gene expression pattern of bone related proteins (osteonectin and osteocalin) in cctopic bone induced by murine BMP4 is comparable to that in embryonic osteogenesis 5. In vitro, BMP2 converts the differentiation pathway of C2C12 myoblasts into osteoblast lineages s, and induces ST2 stromal cells to express a high level of osteoblast p h e n o types, including elevated A K P activity, P T H / P T H r P receptors and osteocalcin production 25. Transfection of the mesenchymal cell lines C3HIOT1/2 with BMP2 or 4 eDNA also induces differ-
entiation into osteogenetic lineage I. BMP2 can cause the differentiation of C3H10T1/2 cells into chondrocytes and osteoblasts at high concentrations 22. These studies indicate that BMP plays an important role in regulating the differentiation pathway of mesenchymal cells into osteoblast lineage cells. MtmD~"~4 suggests that BMP stimulates osteoblast differentiation by enhancing expression of markers such as alkaline phosphatase and osteocalcin as well as the formation of mineralized bone nodules in vitro. The recent identification of T G F fl activity further suggests a possible role for TGF fl in bone formation. T G F fl has been shown to promote bone formation and to enhance bone healing. In vitro, TGF fl may stimulate chemotaxis of osteoblasts 11. T G F fl has a strong proliferative effect on osteoblast precursors and can enhance chondrogenesis in periosteal explants 13 and stimulate chondrogenesis of periosteum-derived cells 6. In vivo, local injection of TGF fl initiates subperioste-
Table 1. Area of newly formed bone induced by rhBMP2/TGF fl and rhBMP2 alone by day 14
No.
rhBMP2/TGF fl rhBMP2 group (nln'l 2) group (ran12)
1 2 3
0~354 0.191 0.124
0.030 0.017 0.042
Average
0.223
0.030
Two-sample analysis (Statgraph software). rhBMP2/TGF fl group >rhBMP2 group, P<0.05.
New bone formation induced by rhBMP2 and TGF ]/ al chondrogenesis and osteogenesis7 and increases the strength of tibial fractures 15. Bone ingrowth is also enhanced by T G F ]/12~ All these facts imply its key role in skeletal growth and remodelling. The difference between these two proteins is that T G F fl lacks an ability to induce ectopic bone formation by itself~7'24. The target cells of BMP are undifferentiated mesenchymal cells, but BMP has no effects on differentiated chondrocytes and osteoblasts. KOMAKI et al. 1~ have shown that BMP2 stimulates the differentiation of both osteoblast and chondrobtast progenitors, but does not change the fate of the respective progenitors. In contrast to BMP, T G F ]/has been viewed as a growth-stimulatory factor for mesenchymal cells. T G F ] / h a s powerful proliferative effects on osteoblasts and chondrocytes. It also stimulates matrix formation and inhibits matrix degradation. REDD117 suggests that BMP acts at an earlier control point at a primordial stage of the initial osteoblast-lineage, while T G F ]/ may regulate the later steps in bone-cartilage lineage. BMP and T G F ]/have been purified from bovine cortical bone and have been shown to promote bone formation in vivo. However, the exact roles that these proteins play during osteogenesis is not well understood. OGAWA et a l ) 6 have shown that implants containing a combination of BMP and T G F ]/exhibit an abundance of cartilagious structures. Histomorphometry is an efficient method to evaluate the formation of new bone21. The morphometrical result of the present study revealed that T G F ]/ alone did not promote ectopic bone formation, while rhBMP2 alone gave rise to a low level of bone and cartilage formation. The combination of rhBMP2 and T G F ]/, however, demonstrated more and well developed bone. These results suggest that bovine T G F ] / e n hanced formation of ectopic bone in mice muscle, when implanted in combination with rhBMP2 in a ceramic carrier. When rhBMP2/TGF ]//CBB was implanted into muscle pouches, rhBMP2 induced the differentiation of mesenchymal cells around the blood vessels into osteoblasts and chondrocytes. Subsequently, T G F ]/stimulated these differentiated ceils to proliferate and synthesize the extracellular matrix proteins characteristic of bone and
cartilage. Because of their different aspects on different stages of cells, they could be synergistic in promoting bone formation.
Acknowledgments. We are grateful to Mr Yu Zhao for technical assistance and Dr Lianjun Yang for typing the manuscript. This work was supported by the National Natural Sciences Foundation of China, No. 39300148.
References 1. AHRENS M, ANKENBAUERT, ScrmODER D, HOLLNAGELA, MAV~ H, GROSSG. Expression of human bone morphogenetic protein-2 or -4 in murine mesenchymal progenitor C3H10T1/2 cells induces differentiation into distinct mesenchymal cell lineages. DNA Cell Biol 1993: 12: 871-80. 2. ASSOIANRK, KOMORIYAA, MEYERSCA, MILLER DM, SVORNMB. Transforming growth factor fl in human platelets: identification of a major storage site, purification and characterization. J Biol Chem 1983: 258: 7155-60. 3. BAYLINKDJ, FINKELMANRD, MOHANS. Growth factors to stimulate bone formation. J Bone Miner Res 1993:8 (Suppl 2): $565-72. 4. CELESTEAJ, SONGJJ, Cox K, ROSENV, WOZNEYJM. Bone morphogenetic protein-9, a new member of the TGF fl superfamily. J Bone Miner Res 1994:9 (Suppl 1): $136. 5. HmOTAS, TAKAOKAK, HASmMOTOJ, et al. Expression of mRNA of routine bonerelated proteins in ectopic bone induced by murine bone morphogenetic protein4. Cell Tissue Res 1994: 277: 27-32. 6. I w ~ M, NAKAHARAH, NAKATAK, NAKASET, KIMURAT, ONO K. Regulation of proliferation and osteoehondrogenie differentiation of periosteum-derived cells by Transforming Growth Factor fl and basic Fibroblast Growth Factor. J Bone Joint Surg 1995: 77A: 543-54. 7. JOYCE M, ROBERTSA, SPORN MB, BOLANDERME. Transforming growth factor fl and the initiation of chondrogenesis and osteogenesis in the rat femur. J Cell Biol 1990: 110: 2195-270. 8. KATAGIRIEA, YAr~KUCHIA, KoMArd M, et al. Bone morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into osteoblast lineage. J Cell Biol 1994: 127: 1755-66. 9. KaNGSEYDM. The TGF fl superfamily: new members, new receptors, and new genetic tests of function in different organisms. Genes Dev 1994: 8: 133-46. 10. KOMAraM, KATAKIRIT, SoDA T Bone morphogenetic protein-2 does not alter the differentiation pathway of committed progenitors of osteoblasts and chon-
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droblasts. Cell Tissue Res 1996: 21M: 917. 11. LIND M, DELEURANB, THESTRuP-PEDERSEN K, SOEBALLE K, ERmSEN EE BLENGERC. Chemotaxis of human osteoblasts: effects on osteotropic growth factors. APMIS 1995: 103: 140-6. 12. LIU L, ZHAOM, WANGHX, et al. Induction of bone formation by recombinant human BMP2 in mice, rats and rabbits. J Bone Joint Surg 1996: 78B (Suppl 1): $25. 13. MIURA Y, FITZStMMONSJS, COMMISSO CN, GALLAYSH, O'DRISCOLLSW. Enhancement of periosteal chondrogenesis in vitro: dose-response for TGF ill. Clin Orthop 1994: 301: 271-80. 14. ML~DY GR. Local control of bone formation by osteoblasts. Clin Orthop 1995: 313: 19-26. 15. NIELSENHM, ANDREASSENTT, LEDETT, OXLUNDH. Local injection of TGF fl increases the strength of tibial fractures in the rat. Acta Orthop Scand 1994: 65: 3741. 16. OGAWAY, SCHMIDTDK, NATHANRM, et al. Bovine bone activin enhances bone morphogenetic protein-induced ectopic bone formation. J Biol Chem 1992: 267: 14233-7. 17. REDDI AH. Bone morphogenetic proteins, bone marrow stromal cells, and mesenchymal stem cells. Clin Orthop 1995: 313: 115-9. 18. ROBERTSAB, SPORNMB, ASSOIANRK, et al. Transforming growth factor p: rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen 9formation in vivo. Proc Natl Acad Sci USA 1986: 83: 4167-71. 19. SEYED~SM, THOMASTC, THOMPSONAY, Ros~N DM, Pmz KA. Purification and characterization of two cartilage-inducing factors from bovine demineralized bone. Proc Natl Acad Sci USA 1985: 82: 2267-71. 20. StrmcEt DR, "Ibx~rERTM, Ptmcmo AF, GOMBOTZ WR, URBAN RM, GALANTE JO. Enhancement of bone ingrowth by transforming growth factor ft. J Bone Joint Surg 1995: 77A: 1135-47. 21. VUAANENV~, LINDHOLMTC, GAO TJ, LINOHOLM TS. Low dosage of native allogeneic bone morphogenetic protein in repair of sheep calvarial defects. Int J Oral Maxillofac Surg 1997: 26: 38993. 22. WANGEA, ISRAELDI, KELLYS, LUXENBERGDP. Bone morphogenetic protein-2 cause commitment and differentiation in C3H10T1/2 and 3T3 cells. Growth Factors 1993: 9: 57-71. 23. WANGEA, ROSENV, D'ALESSANDROJS, et al. Recombinant human bone morphogenetic protein induces bone formation. Proc Natl Acad Sci USA 1990: 87: 22204. 24. YAMAGUCHIA. Regulation of differentiation pathway of skeletal mesenchymal cells in cell lines by transforming growth
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factor fl superfamily. Seminars in Cell Biol 1995: 6: 165-73. 25. YAMAGUCmA, IS~ZUYA T, ICrm~u N, et al. Effects of BMP-2, BMP-4 and BMP-
6 on osteoblastic differentiation of bone marrow-derived stromal cell lines, ST2 and MC3T3-G2/PA6. Biochem Biophys Commun Res 1996: 220: 366-71.
Address:
Professor Yah Jin Department of Oral Pathology ~ Stomatological College Fourth Military Medical University Xi'an 710032 PR China Tel: +86 29 2527961 (H) E-mail: [email protected]
Int. J. Oral Maxillofac. Surg. 1998; 27." 310-314 Printed in Denmark. All rights reserved
lnternan'onalJournal of
Ord & Maxillo[acidSurgery ISSN 0901-5027
Induction of new bone by ceramic bovine bonewith recombinant human bone morphogenetic protein 2 and transforming growth factor/
Xlaohui Si, Yan Jin, Llanjla Yang Department of Oral Pathology, Stomatological College, Fourth Military Medical University, Xi'an, PR China
X. Si, Y. Jin, L. Yang: Induction of new bone by ceramic bovine bone with recombinant human bone morphogenetic protein 2 and transforming growth factor ft. Int. J. Oral Maxillofac. Surg. 1998; 27: 310-314. 9 Munksgaard, 1998 Abstract. Bone mt)rphogenetic protein (BMP) has the ability to induce ectopic bone, while the action of transforming growth factor fl (TGF t5) is to stimulate proliferation of osteoblasts and chondrocytes as well as the production of extracellular matrix. The aim of the present study was to study their synergistic actions in bone formation. Three kinds of complexes, recombinant human BMP2 (rhBMP2), T G F fl and rhBMP2frGF fl in ceramic bovine bone (CBB), were made and then implanted into the thigh muscle pouches of mice. The histological reactions of the implanted areas were studied at intervals of 3, 5, 7, 14 and 21 days. The results showed that, except for the implants with T G F fl alone, both rhBMP2 and rhBMP2/TGF fl implants exhibited new ectopic bone formation. The morphometric study revealed that the quantity of newly formed bone induced by rhBMP2/TGF fl was obviously greater than by rhBMP2 alone. These results indicate that T G F fl in combination with BMP may enhance formation of ectopic bone.
A variety of bone growth factors play a role in bone morphogenesis, development, growth, repair and remodelling. It is apparent that bone contains a number of structurally related proteins, including bone morphogenetic protein (BMP) and transforming growth factor fl (TGF fl), that are involved in bone formation 3. BMP and T G F fl belong to the T G F fl superfamily. This family is composed of at least 25 members of the molecules which retain the seven cysteine residues within the mature region of the protein 9,24. Of these, BMP and T G F fl are receiving considerable attention for potential clinical application. BMP is a protein which can induce new
cartilage and bone formation in extraskeletal tissues by stimulating undifferentiated mesenchymal cells to proliferate and differentiate into bone-forming cells. Human cDNA of BMP1 and eight other related BMPs (BMP2BMP9) have been cloned and expressed 4,23. Recombinant human BMP2 (rhBMP2) is considered to be the most active and could induce new bone formation at both bony and nonbony sites alone. T G F fl is present in most tissues and is known to play an important regulatory role in cell growth, migration and differentiation. Platelets are a major storage site for this growth factor, and they contain 10-100-fold more
Key words: bone morphogeneUc protein; transforming growth factor 13;bone induction; ceramic bovine bone. Accepted for publication 30 December 1997
T G F fl than do the other tissues 2. T G F fl has been shown to promote bone formation and to enhance bone healing, and is present in bone in high quantities 19. In vitro and in vivo studies have indicated that T G F ] / h a s stimulatory effects on proliferation and extracellular matrix synthesis in many types of skeletal tissue cells. BMP and T G F fl may be synergistic in promoting bone formation because of their effects on different stages of cells. The combination of BMP and T G F fl might result in better bone growth than each of them separately. Thus, the aim of the present study was to observe the BMPbone induction influenced by T G F ft.
New bone formation induced by r h B M P 2 and TGF fl Materials and methods Growth factors
TGF fl was obtained from fresh bovine blood platetets by acid/ethanol procedure and chromatography. The bioassay of TGF fl determined the ability of the polypeptide to induce anchorage-independent growth in nonneoplastic NRK fibroblasts by measuring the formation of colonies of cells in soft agar2. An in vivo study of TGF fl was performed as described earlierIs and it showed that TGF fl could cause the formation of granulation tissues at the site of injection in newborn mice. rhBMP2 is the purified recombinant protein from E. coli, and was kindly provided by Dr M. Zhao (Institute of Basic Medical Science, Academy of Military Medical Science, PR China). The in vivo ectopic bone formation assay revealed that this material was active at 0.5 rag, when tested seven days after implantation12.
Complex preparation
The blocks of multipore ceramic bovine bone (CBB) were produced from fresh bovine rib bones which consist of a cortical layer and a cancellous part. The resected ribs were prepared by boiling them and cleaning them from the periosteum and adjacent connective tissues and then cut into blocks. The blocks were washed in normal saline, soaked in H202 to remove proteins and then washed in running water. The deproteinized blocks were heated at 900~ for 1 h. Each block weighed about 20 mg and was selected as the carrier for combining rhBMP2 and/or T G F ft. Three kinds of complexes were made: rhBMP2 and CBB (rhBMP2/CBB), CBB and the purified bovine T G F fl ( T G F fl/CBB), and rhBMP2/TGF fl/
311
CBB. The CBB implants were coated with rhBMP2 and/or T G F fl under aseptic conditions by soaking them in solutions containing rhBMP2 and/or T G F fl for 18 hours at 4~ and then lyophilized. The dose of T G F fl and rhBMP2 for combination with each CBB block were 15 ng and 2 mg respectively. Animal procedures and histological examination
A total of 45 mice were randomly divided into three experimental groups for the implantation of rhBMP2/CBB (Group 1), TGF/CBB (Group 2), and rhBMP2/TGF/CBB (Group 3). The mice were anesthetized with ether and the above complexes were implanted into their right thigh muscle pouches. Each complex was implanted into 15
Fig. 1. rhBMP2/TGF fl/CBB implantation (day 3). ThiS section shows aggregation of mesenchymal cells in muscle (HEX400).
Fig. 2. rhBMP2/rGF fl/CBB implantation (day 5). Proliferation of mesenchymal cells is visible (HEX400).
Fig. 3. rhBMP2/TGF fl/CBB implantation (day 7). Mesenchymal cells differentiated into chondrocytes (HE•
Fig. 4. rhBMP2/TGF fl/CBB implantation (day 14). Newly formed bone is seen (HEx200).
312
Si et al.
Fig. 5. rhBMP2/CBB implantation (day 14). rhBMP2 alone exhibited
low levels of woven bone (HE•
animals. The animals were killed at intervals of 3, 5, 7, 14 and 21 days after implantation. Tissues were fixed for 24 h in 10% formalin, followed by decalcification in formic acid/formalin for 72 h. Paraffin-embedded specimens were stained with hematoxylin and eosin. The area of newly formed bone by day 14 was measured-by using the lattice in the eye-piece of the microscope (10x). The data were analysed by using Statgraph software. Results
There was no new bone formation in group 2, i.e. TGFfl/CBB blocks. Proliferation of fibroblasts was seen by day 3 and day 5 and a thin condensed layer of fibrous connective tissue, surrounding the implanted blocks, by day 21. Both group 1 and group 3 showed aggregation and proliferation of round and spindle-shape mesenchymal cells by day 3 and day 5 (Figs. 1, 2). There was mild inflammatory cell infiltration in the implanted areas. By comparison, the inflammatory reaction was less in group 3 than in group 1 five days postimplantation. The inflammatory reaction disappeared by day 7 and both showed formation of new cartilage (Fig. 3) and woven bone with newly formed marrow by day 14 (Figs. 4, 5). The newly formed bone trabeculac were thicker in ,group 3 than in group 1. The newly formed bone marrow of group 3 appeared a few days earlier than in group 1. By day 21, newly formed bone became mature and cortical bone with bone marrow was observed (Fig. 6). Newly
Fig. 6. rhBMP2/TGF fl/CBB implantation (day 21). Mature cortical bone has formed (HE•
formed bone tissue was detected throughout the full thickness of the implants of rhBMP2/CBB and rhBMP2/TGF fl/CBB. A delicate reticular network of newly formed bone was seen in the pores of the central area of the CBB. A morphometric study revealed that the average area of newly formed bone by day 14 in group 3 was 0.223 mm 2, whereas in group 1 it was 0.030 mm 2. Analysis showed the difference between group 3 and group 1 to be statistically significant (t-test, Table 1). Discussion
BMP irreversibly induces differentiation of perivascular mesenchymaltype cells into cartilage and bone-forming cells. It is capable of inducing ectopic bone formation in vivo. Low dosage of native allogeneic BMP showed strong osteoinductivity and low immunogenicity21. The sequence of cctopic bone formation induced by BMP is similar to that of embryonic bone formarion and fracture repair. The sequential gene expression pattern of bone related proteins (osteonectin and osteocalin) in cctopic bone induced by murine BMP4 is comparable to that in embryonic osteogenesis 5. In vitro, BMP2 converts the differentiation pathway of C2C12 myoblasts into osteoblast lineages s, and induces ST2 stromal cells to express a high level of osteoblast p h e n o types, including elevated A K P activity, P T H / P T H r P receptors and osteocalcin production 25. Transfection of the mesenchymal cell lines C3HIOT1/2 with BMP2 or 4 eDNA also induces differ-
entiation into osteogenetic lineage I. BMP2 can cause the differentiation of C3H10T1/2 cells into chondrocytes and osteoblasts at high concentrations 22. These studies indicate that BMP plays an important role in regulating the differentiation pathway of mesenchymal cells into osteoblast lineage cells. MtmD~"~4 suggests that BMP stimulates osteoblast differentiation by enhancing expression of markers such as alkaline phosphatase and osteocalcin as well as the formation of mineralized bone nodules in vitro. The recent identification of T G F fl activity further suggests a possible role for TGF fl in bone formation. T G F fl has been shown to promote bone formation and to enhance bone healing. In vitro, TGF fl may stimulate chemotaxis of osteoblasts 11. T G F fl has a strong proliferative effect on osteoblast precursors and can enhance chondrogenesis in periosteal explants 13 and stimulate chondrogenesis of periosteum-derived cells 6. In vivo, local injection of TGF fl initiates subperioste-
Table 1. Area of newly formed bone induced by rhBMP2/TGF fl and rhBMP2 alone by day 14
No.
rhBMP2/TGF fl rhBMP2 group (nln'l 2) group (ran12)
1 2 3
0~354 0.191 0.124
0.030 0.017 0.042
Average
0.223
0.030
Two-sample analysis (Statgraph software). rhBMP2/TGF fl group >rhBMP2 group, P<0.05.
New bone formation induced by rhBMP2 and TGF ]/ al chondrogenesis and osteogenesis7 and increases the strength of tibial fractures 15. Bone ingrowth is also enhanced by T G F ]/12~ All these facts imply its key role in skeletal growth and remodelling. The difference between these two proteins is that T G F fl lacks an ability to induce ectopic bone formation by itself~7'24. The target cells of BMP are undifferentiated mesenchymal cells, but BMP has no effects on differentiated chondrocytes and osteoblasts. KOMAKI et al. 1~ have shown that BMP2 stimulates the differentiation of both osteoblast and chondrobtast progenitors, but does not change the fate of the respective progenitors. In contrast to BMP, T G F ]/has been viewed as a growth-stimulatory factor for mesenchymal cells. T G F ] / h a s powerful proliferative effects on osteoblasts and chondrocytes. It also stimulates matrix formation and inhibits matrix degradation. REDD117 suggests that BMP acts at an earlier control point at a primordial stage of the initial osteoblast-lineage, while T G F ]/ may regulate the later steps in bone-cartilage lineage. BMP and T G F ]/have been purified from bovine cortical bone and have been shown to promote bone formation in vivo. However, the exact roles that these proteins play during osteogenesis is not well understood. OGAWA et a l ) 6 have shown that implants containing a combination of BMP and T G F ]/exhibit an abundance of cartilagious structures. Histomorphometry is an efficient method to evaluate the formation of new bone21. The morphometrical result of the present study revealed that T G F ]/ alone did not promote ectopic bone formation, while rhBMP2 alone gave rise to a low level of bone and cartilage formation. The combination of rhBMP2 and T G F ]/, however, demonstrated more and well developed bone. These results suggest that bovine T G F ] / e n hanced formation of ectopic bone in mice muscle, when implanted in combination with rhBMP2 in a ceramic carrier. When rhBMP2/TGF ]//CBB was implanted into muscle pouches, rhBMP2 induced the differentiation of mesenchymal cells around the blood vessels into osteoblasts and chondrocytes. Subsequently, T G F ]/stimulated these differentiated ceils to proliferate and synthesize the extracellular matrix proteins characteristic of bone and
cartilage. Because of their different aspects on different stages of cells, they could be synergistic in promoting bone formation.
Acknowledgments. We are grateful to Mr Yu Zhao for technical assistance and Dr Lianjun Yang for typing the manuscript. This work was supported by the National Natural Sciences Foundation of China, No. 39300148.
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Address:
Professor Yah Jin Department of Oral Pathology ~ Stomatological College Fourth Military Medical University Xi'an 710032 PR China Tel: +86 29 2527961 (H) E-mail: [email protected]