An adhesive bone marrow scaffold and bone morphogenetic-2 protein carrier for cartilage tissue engineering

Abstract

A chondroitin sulfate-bone marrow (CS-BM) adhesive hydrogel was used to localize rhBMP-2 to enhance articular cartilage tissue formation. Chondrocyte pellet culture revealed that 0.1 and 1 μg/mL of rhBMP-2 enhanced sulfated-GAG content. rhBMP-2 localization within the hydrogels was investigated, and it was found that BM, CS-NHS, and rhBMP-2 levels and time affected rhBMP-2 retention. Retention was modulated from 82 to 99% over a 3-week period for the material formulations investigated. To evaluate carrier efficacy, rhBMP-2 and bovine articular chondrocytes were encapsulated within CS-BM, and biochemical evaluation revealed significant increases in total collagen production with rhBMP-2. Histological analysis revealed more robust tissue formation and greater type-II collagen production with encapsulated rhBMP-2. Subsequently, a subcutaneous culture of hydrogels revealed increased total collagen, type-II to type-I collagen ratio, and sulfated GAG in samples carrying rhBMP-2. These findings indicate the development of a multifunctional system capable of localizing rhBMP-2 to enhance repair tissue quality.

Fig 1.

(A) Dry weight, (B) sulfated GAG mass fraction, and (C) safranin-O staining reveal quantitative and qualitative differences among chondrocyte pellets cultured in various rhBMP-2 conditions. Scale bar = 500 μm. * P < 0.05, ** P < 0.01 *** P < 0.005. For (A) and (B), significant differences refer to time points 6 and 10 days.

Fig 2.

(A) Schematic of chondrocytes and rhBMP-2 encapsulated within a CS-BM hydrogel. (B) Hydroxyproline normalized to chondrocyte DNA at 21 days of In Vitro hydrogel culture. * P < 0.05, ** P <0.01, *** P < 0.005.

Fig 3.

Histology and immunohistochemistry of chondrocyte encapsulated hydrogels after 21 days of In Vitro culture. (A) Safranin-O and (B) type-II collagen immunohistochemistry were performed to qualitatively assess cell phenotype and hyaline nature of regenerated tissue. Scale bar = 100 μm.

Fig 4.

Biochemistry assays for bovine chondrocytes encapsulated in subcutaneously implanted hydrogels. (A) Hydroxyproline assay was performed at both the three and six-week time points to assess the effect of rhBMP-2 loading on total collagen production. (B) Dimethyl-methylene blue assay was performed at the six- week time point to quantify the effect of rhBMP-2 on sulfated GAG production.

Fig 5.

Immunohistochemistry of subcutaneously implanted CSBM hydrogels containing encapsulated bovine chondrocytes. (A) Low and high magnification images of subcutaneously implanted constructs stained with antibodies against type-I and type-II collagen (scale bars equal 1 mm and 100 μm, respectively). Analysis of the stained neocartilage using a thresholding and fraction of maximum intensity method to assess the effect of rhBMP-2 on (B) the percent of tissue stained positive for type-II collagen and (C) the relative ratio of type-II to type-I collagen. * P < 0.05