Search tips
Search criteria 


Logo of aapspharmspringer.comThis journalToc AlertsSubmit OnlineOpen Choice
AAPS PharmSciTech. 2001 September; 2(3): 73–80.
Published online 2001 October 7. doi:  10.1208/pt020318
PMCID: PMC2750583

Effect of a freeze-dried CMC/PLGA microsphere matrix of rhBMP-2 on bone healing


The hypothesis of this research was that implants of poly(lactide-co-glycolide) (PLGA) microspheres loaded with bone morphogenetic protein-2 (rhBMP-2) and distributed in a freeze-dried carboxymethylcellulose (CMC) matrix would produce more new bone than would matrix implants of non-protein-loaded microspheres or matrix implants of only CMC. To test this hypothesis it was necessary to fashion microsphere-loaded CMC implants that were simple to insert, fit precisely into a defect, and would not elicit swelling. Microspheres were produced via a water-in-oil-in-water double-emulsion system and were loaded with rhBMP-2 by soaking them in a buffered solution of the protein at a concentration of 5.4 mg protein per gram of PLGA. Following recovery of the loaded microspheres by lyophilization matrices for implantation were prepared by lyophilizing a suspension of the microspheres in 2% CMC in flat-bottom tissue culture plates. Similar matrices were made with 2% CMC and with 2% CMC containing blank microspheres. A full-thickness calvarial defect model in New Zealand white rabbits was used to assess bone growth. Implants fit the defect well allowing for direct application. Six weeks postsurgery, defects were collected and processed for undecalcified histology. In vitro, 60% of the loaded rhBMP-2 released from devices or microspheres in 5 to 7 days. With the unembedded microspheres releasing faster than those embedded in CMC In vivo. the rhBMP-2 microspheres greatly enhanced bone healing, whereas nonloaded PLGA microspheres in the CMC implants had little effect. The results showed that a lyophilized device of rhBMP-2 PLGA microspheres in CMC was an effective implantable protein-delivery system for the use in bone repair.

Keywords: bone morphogenetic protein-2, PLGA microspheres, controlled delivery, protein delivery, in vitro, in vivo, bone repair

Full Text

The Full Text of this article is available as a PDF (402K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
1. Alonso MJ, Cohen S, Park TG, Gupta RK, Siber GR, Langer R. Determinants of release rate of tetanus vaceine from polyester microspheres. Pharm Res. 1993;10:945–953. doi: 10.1023/A:1018942118148. [PubMed] [Cross Ref]
2. Johnson OL, Cleland JL, Lee HJ, et al. A monthlong effect from a single injection of microencapsulated human growth hormone. Nat Med. 1996;2:795–799. doi: 10.1038/nm0796-795. [PubMed] [Cross Ref]
3. Okada H, Doken Y, Ogawa Y, Toguchi H. Preparation of three-month depot injectable microspheres of leuprorelin acetate using biodegradable polymers. Pharm Res. 1994;11:1143–1147. doi: 10.1023/A:1018936815654. [PubMed] [Cross Ref]
4. Wang EA, Israel DI, Kelly S, Luxenberg DP. Bone morphogenetic protein-2 causes commitment and differentiation in C3H10T1/2 and 3T3 cells. Growth Factors. 1993;9:57–71. doi: 10.3109/08977199308991582. [PubMed] [Cross Ref]
5. Wozney JM, Rosen V, Celeste AJ, et al. Novel regulators of bone formation: molecular clones and activities. Science. 1988;242:1528–1534. doi: 10.1126/science.3201241. [PubMed] [Cross Ref]
6. Luyten FP, Cunningham NS, Ma S, et al. Puritication and partial amino acid sequence of osteogenin, a protein initiating bone differentiation. J Biol Chem. 1989;264:13377–13380. [PubMed]
7. Celeste AJ, Jannazzi JA, Taylor RC, et al. Identification of transforming growth factor beta family members present in bone-inductive protein purified from bovine bone. Proc Natl Acad Sci U.S.A. 1990;87:9843–9847. doi: 10.1073/pnas.87.24.9843. [PubMed] [Cross Ref]
8. Ozkaynak E, Rueger DC, Drier EA, et al. OP-1 cDNA encodes an osteogenic protein in the TGF-beta family. Embo J. 1990;9:2085–2093. [PubMed]
9. Sampath TK, Coughlin JE, Whetstone RM, et al. Bovine osteogenic protein is composed of dimers of OP-1 and BMP-2A, two members of the transforming growth factor-beta superfamily. J Biol Chem. 1990;265:13198–13205. [PubMed]
10. Ozkaynak E, Schnegelsberg PN, Jin DF, et al. Osteogenic protein-2: a new member of the transforming growth factor-beta superfamily expressed early in embryogenesis. J Biol Che. 1992;267:25220–25227. [PubMed]
11. Rosen V, Thies RS. The BMP proteins in bone formation and repair. Trends Genet. 1992;8:97–102. [PubMed]
12. Kenley RA, Yim K, Abrams J, et al. Biotechnology and bone graft substitutes. Pharm Res. 1993;10:1393–1401. doi: 10.1023/A:1018902720816. [PubMed] [Cross Ref]
13. Lynch SE, Buser D, Hernandez RA, et al. Effects of the platelet-derived growth factor/insulin-like growth factor-I combination on bone regeneration around titanium dental implants: results of a pilot study in beagle dogs. J Periodontol. 1991;62:710–716. [PubMed]
14. Kenley R, Marden L, Turek T, Jin L, Ron E, Hollinger JO. Osseous regeneration in the rat calvarium using novel delivery systems for recombinant human bone morphogenetic protein-2 (rhBMP-2) J Biomed Mater Res. 1994;28:1139–1147. doi: 10.1002/jbm.820281004. [PubMed] [Cross Ref]
15. Mayer M, Hollinger J, Ron E, Wozney J. Maxillary alveolar cleft repair in dogs using recombinant human bone morphogenetic protein-2 and a polymer carrier [see comments] Plast Reconstr Surg. 1996;98:247–259. doi: 10.1097/00006534-199608000-00006. [PubMed] [Cross Ref]
16. Duggirala SS, Rodgers JB, DeLuca PP. The evaluation of lyophilized polymer matrices for administering recombinant human bone morphogenetic protein-2 [published erratum appears in Pharm Dev Technol. 1996;1(3):317] Pharm Dev Technol. 1996;1:165–174. doi: 10.3109/10837459609029891. [PubMed] [Cross Ref]
17. Toriumi DM, Kotler HS, Luxenberg DP, Holtrop ME, Wang EA. Mandibular reconstruction with a recombinant bone-inducing factor: functional. histologic, and biomechanical evaluation. Arch Otolaryngol Head Neck Surg. 1991;117:1101–1112. [PubMed]
18. Yasko AW, Lane JM, Fellinger EJ, Rosen V, Wozney JM, Wang EA. The healing of segmental bone defects. induced by recombinant human bone morphogenetic protein (rhBMP-2). A radiographic. histological, and biomechanical study in rats[published erratum appears in J Bone Joint Surg Am. 1992:74(7):1111] J Bone Joint Surg Am. 1992;74:659–670. [PubMed]
19. Gerhart TN, Kirker-Head CA, Kriz MJ, et al Healing segmental femoral defects in sheep using recombinant human bone morphogenetic protein. Clin Orthop. 1993;317–326. [PubMed]
20. Cook SD, Baffes GC, Wolfe MW, Sampath TK, Rueger DC. Whitecloud TS 3rd. The effect of recombinant human osteogenic protein-1 on healing of large segmental bone defects. J Bone Joint Surg Am. 1994;76:827–838. [PubMed]
21. Marden LJ, Hollinger JO, Chaudhari A, Turek T, Schaub RG, Ron E. Recombinant human bone morphogenetic protein-2 is superior to demineralized bone matrix in repairing craniotomy defects in rats. J Biomed Mater Res. 1994;28:1127–1138. doi: 10.1002/jbm.820281003. [PubMed] [Cross Ref]
22. Urist MR, O'Connor BT, Burwell RG. Bone grafts, derivatives, and substitutes. Oxford, UK: Butterworth-Heinemann; 1994.
23. Lucas PA, Syftestad GT, Goldberg VM, Caplan AI. Ectopic induction of cartilage and bone by water-soluble proteins from bovine bone using a collagenous delivery vehicle. J Biomed Mater Res. 1989;23:23–39. doi: 10.1002/jbm.820231306. [PubMed] [Cross Ref]
24. Duggirala SS, Mehta RC, DeLuca PP. Interaction of recombinant human bone morphogenetic protein-2 with poly(d,l lactide-co-glycolide) microspheres. Pharm Dev Technol. 1996;1:11–19. doi: 10.3109/10837459609031413. [PubMed] [Cross Ref]
25. Schrier JA, DeLuca PP. Recombinant human bone morphogenetic protein-2 binding and incorporation in PLGA microsphere delivery systems. Pharm Dev Technol. 1999;4:611–621. doi: 10.1081/PDT-100101400. [PubMed] [Cross Ref]
26. Rodgers JB, Vasconez HC, Wells MD, et al. Two lyophilized polymer matrix recombinant human bone morphogenetic protein-2 carriers in rabbit calvarial defects. J Craniofac Surg. 1998;9:147–153. doi: 10.1097/00001665-199803000-00012. [PubMed] [Cross Ref]
27. Hausberger AG, DeLuca PP. Characterization of biodegradable poly(D,L-lactide-co-glycolide) polymers and microspheres. J Pharm Biomed Anal. 1995;13:747–760. doi: 10.1016/0731-7085(95)01276-Q. [PubMed] [Cross Ref]
28. Isobe M, Yamazaki Y, Mori M, Ishihara K, Nakabayashi N, Amagasa T. The role of recombinant human bone morphogenetic protein-2 in PLGA capsules at an extraskeletal site of the rat. J Biomed Mater Res. 1999;45:36–41. doi: 10.1002/(SICI)1097-4636(199904)45:1<36::AID-JBM5>3.0.CO;2-I. [PubMed] [Cross Ref]
29. Hollinger JO, Schmitt JM, Buck DC, et al. Recombinant human bone morphogenetic protein-2 and collagen for bone regeneration. J Biomed Mater Res. 1998;43:356–364. doi: 10.1002/(SICI)1097-4636(199824)43:4<356::AID-JBM3>3.0.CO;2-7. [PubMed] [Cross Ref]
30. Brekke JH, Toth JM. Principles of tissue engineering applied to programmable osteogenesis. J Biomed Mater Res. 1998;43:380–398. doi: 10.1002/(SICI)1097-4636(199824)43:4<380::AID-JBM6>3.0.CO;2-D. [PubMed] [Cross Ref]
31. Schrier JA, DeLuca PP. Porous bone morphogenetic protein 2 microspheres polymer binding and in vitro release.AAPS Pharm Sci Tech 2001:2(3) Article 17. Available from: pharmscitech/volume2issue3/1103/manuscript.htm [PMC free article] [PubMed]

Articles from AAPS PharmSciTech are provided here courtesy of American Association of Pharmaceutical Scientists