FTIR-I compositional mapping of the cartilage-to-bone interface as a function of tissue region and age

Abstract

Soft tissue-to-bone transitions, such as the osteochondral interface, are complex junctions that connect multiple tissue types and are critical for musculoskeletal function. The osteochondral interface enables pressurization of articular cartilage, facilitates load transfer between cartilage and bone, and serves as a barrier between these two distinct tissues. Presently, there is a lack of quantitative understanding of the matrix and mineral distribution across this multitissue transition. Moreover, age-related changes at the interface with the onset of skeletal maturity are also not well understood. Therefore, the objective of this study is to characterize the cartilage-to-bone transition as a function of age, using Fourier transform infrared spectroscopic imaging (FTIR-I) analysis to map region-dependent changes in collagen, proteoglycan, and mineral distribution, as well as collagen organization. Both tissue-dependent and age-related changes were observed, underscoring the role of postnatal physiological loading in matrix remodeling. It was observed that the relative collagen content increased continuously from cartilage to bone, whereas proteoglycan peaked within the deep zone of cartilage. With age, collagen content across the interface increased, accompanied by a higher degree of collagen alignment in both the surface and deep zone cartilage. Interestingly, regardless of age, mineral content increased exponentially across the calcified cartilage interface. These observations reveal new insights into both region- and age-dependent changes across the cartilage-to-bone junction and will serve as critical benchmark parameters for current efforts in integrative cartilage repair.

Keywords: AGING; BIOENGINEERING; COLLAGEN; MATRIX MINERALIZATION; OSTEOARTHRITIS.

Fig. 1

Cartilage-to-bone junction. Immature and mature osteochondral samples exhibit region-dependent changes in (A) matrix distribution (n = 3, picrosirius red for collagen, alcian blue for proteoglycan, von Kossa for mineral, 5×, scale bar = 200μm). (B) Characteristic IR spectrum for each tissue region (articular cartilage [AC], calcified cartilage [CC], bone) in immature samples. Note Amide I and II (Am I andAmII) peaks are indicative for collagen, the carbohydrate peak for proteoglycan, and phosphate peak for mineral.

Fig. 2

Collagen content, distribution, and organization. Peak integration maps (n = 3) of amide I show relative collagen content and distribution. Line scans across the cartilage-to-bone junction reveal a stepwise increase in collagen content for immature specimens and a gradual increase for mature specimens (articular cartilage [AC], calcified cartilage [CC]). Peak integration maps (n = 3) of the amide I, normalized by amide II, show collagen orientation (blue = perpendicular to polarizer, yellow = mixed orientation, red = parallel to polarizer). Corresponding line scans of collagen orientation show an increase in alignment in the surface zone and deep zone of mature cartilage compared with immature cartilage. Comparison of immature and mature collagen content (n = 3, ^*p < 0.05, differences between zones/regions for immature samples; ^**p < 0.05, differences between zones/regions for mature samples; ^p < 0.05, differences between corresponding immature and mature zones/regions).

Fig. 3

Proteoglycan content and distribution. Peak integration maps (n = 3) of the carbohydrate band and normalized carbohydrate band show relative proteoglycan distribution. Line scans across the cartilage-to-bone junction reveal a peak in non-normalized proteoglycan content in deep zone cartilage for both immature and mature specimens (articular cartilage [AC], calcified cartilage [CC]). Comparison of immature and mature proteoglycan content (n = 3, ^*p < 0.05, differences between zones/regions for immature samples; ^**p < 0.05, differences between zones/regions for mature samples; ^p < 0.05, differences between corresponding immature and mature zones/regions).

Fig. 4

Mineral content and distribution. Peak integration maps (n = 3) of the phosphate band, normalized by amide I, show relative mineral distribution. Peak integration maps (n = 3) of the carbonate band, normalized by amide I, show carbonate distribution. Line scans across the cartilage-to-bone junction reveal a distinct transition from mineral-free to mineral-rich tissue regions for both immature and mature specimens (articular cartilage [AC], calcified cartilage [CC], Am I-Amide I). Comparison of immature and mature mineral content (^*p < 0.05, differences between zones/regions for immature samples; ^**p < 0.05, differences between zones/regions for mature samples; ^p < 0.05, differences between corresponding immature and mature zones/regions).

Fig. 5

Matrix distribution across interface. Results from three different joints (red, blue, and green) are compared for each age group. The normalized phosphate content of the transition region is intermediate between deep zone uncalcified cartilage and bone (^*p < 0.05, difference with uncalcified cartilage; ^**p < 0.05, difference with both uncalcified cartilage and calcified cartilage). Linear regression analyses of region-dependent distribution of matrix components reveal relatively linear fits for collagen and proteoglycan regardless of age, whereas an exponential mineral gradient was observed in immature and mature samples.