title: Biodegradable chitosan scaffolds containing microspheres as carriers for controlled transforming growth factor-beta1 delivery for cartilage tissue engineering.
abstract: BACKGROUND
  Natural articular cartilage has a limited capacity for spontaneous regeneration. Controlled release of transforming growth factor-beta1 (TGF-beta1) to cartilage defects can enhance chondrogenesis. In this study, we assessed the feasibility of using biodegradable chitosan microspheres as carriers for controlled TGF-beta1 delivery and the effect of released TGF-beta1 on the chondrogenic potential of chondrocytes.
  
  
  METHODS
  Chitosan scaffolds and chitosan microspheres loaded with TGF-beta1 were prepared by the freeze-drying and the emulsion-crosslinking method respectively. In vitro drug release kinetics, as measured by enzyme-linked immunosorbent assay, was monitored for 7 days. Lysozyme degradation was performed for 4 weeks to detect in vitro degradability of the scaffolds and the microspheres. Rabbit chondrocytes were seeded on the scaffolds containing TGF-beta1 microspheres and incubated in vitro for 3 weeks. Histological examination and type II collagen immunohistochemical staining was performed to evaluate the effects of released TGF-beta1 on cell adhesivity, proliferation and synthesis of the extracellular matrix.
  
  
  RESULTS
  TGF-beta1 was encapsulated into chitosan microspheres and the encapsulation efficiency of TGF-beta1 was high (90.1%). During 4 weeks of incubation in lysozyme solution for in vitro degradation, the mass of both the scaffolds and the microspheres decreased continuously and significant morphological changes was noticed. From the release experiments, it was found that TGF-beta1 could be released from the microspheres in a multiphasic fashion including an initial burst phase, a slow linear release phase and a plateau phase. The release amount of TGF-beta1 was 37.4%, 50.7%, 61.3%, and 63.5% for 1, 3, 5, and 7 days respectively. At 21 days after cultivation, type II collagen immunohistochemical staining was performed. The mean percentage of positive cells for collagen type II in control group (32.7% +/- 10.4%) was significantly lower than that in the controlled TGF-beta1 release group (92.4% +/- 4.8%, P < 0.05). Both the proliferation rate and production of collagen type II in the transforming growth factor-beta1 microsphere incorporated scaffolds were significantly higher than those in the scaffolds without microspheres, indicating that the activity of TGF-beta1 was retained during microsphere fabrication and after growth factor release.
  
  
  CONCLUSIONS
  Chitosan microspheres can serve as delivery vehicles for controlled release of TGF-beta1, and the released growth factor can augment chondrocytes proliferation and synthesis of extracellular matrix. Chitosan scaffolds incorporated with chitosan microspheres loaded with TGF-beta1 possess a promising potential to be applied for controlled cytokine delivery and cartilage tissue engineering.
authors.affiliation: Department of Orthopedics, Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, China.
authors.forename: Dao-zhang
authors.initials: DZ
authors.lastname: Cai
authors.forename: Chun
authors.initials: C
authors.lastname: Zeng
authors.forename: Da-ping
authors.initials: DP
authors.lastname: Quan
authors.forename: Li-si
authors.initials: LS
authors.lastname: Bu
authors.forename: Kun
authors.initials: K
authors.lastname: Wang
authors.forename: Hua-ding
authors.initials: HD
authors.lastname: Lu
authors.forename: Xiao-feng
authors.initials: XF
authors.lastname: Li
mesh_terms: D000818:Animals; D002356:Cartilage / Q000378:metabolism*; D049109:Cell Proliferation; D048271:Chitosan / Q000008:administration & dosage*; D019902:Chondrocytes / Q000166:cytology; D004337:Drug Carriers; D008863:Microspheres; D011817:Rabbits; D023822:Tissue Engineering / Q000379:methods*; D053773:Transforming Growth Factor beta1 / Q000008:administration & dosage* / Q000737:chemistry
chemical_list: D004337:Drug Carriers; D053773:Transforming Growth Factor beta1; D048271:Chitosan