Evaluation of morphological parameters of bone formation in Sprague–Dawley rats of different ages by in vivo fluorochrome labeling

Figures & data

Table I. Measurements of proximal tibial metaphyseal cancellous bone.

Measured parameters	Abbreviations	Definitions	Unit
Tissue area	T.Ar	Metaphyseal area between 1 and 4 mm distal to the growth plate–epiphyseal junction	mm^2
Trabecular area	Tb.Ar	Total cancellous bone area within total area	mm^2
Trabecular perimeter	Tb.Pm	The perimeter of Tb.Ar	mm
Single label perimeter	sL.Pm	The length of trabecular surface labeled with tetracycline or calcein	mm
Double label perimeter	dL.Pm	The length of trabecular surface labeled with both tetracycline and calcein	mm
Interlabel width	Int.Wi	The distance between tetracycline and calcein labels	μm
Interlabel width (growth plate)	G-Int.Wi	The distance between tetracycline and calcein labels in growth plate metaphyseal junction region	μm

Table II. Static- and dynamic-parameter formulas.

Parameters	Formulas	Units	References
Static parameters
Tb.Ar	Tb.Ar/T.Ar × 100	%	Parfitt et al. (1987)
Tb.Th	(2000/1.199) × (T.Ar/Tb.Pm)	μm	Parfitt et al. (1987)
Tb.N	(1.199/2) × (Tb.Pm/T.Ar)	No./mm	Parfitt et al. (1987)
Tb.Sp	(2000/1.199) × (T.Ar-Tb.Ar)/Tb.Pm	μm	Parfitt et al. (1987)
Dynamic parameters
%L.Pm	(dL.Pm+sL.Pm/2)/Tb.Pm × 100	%	Parfitt et al. (1987)
MAR	Int.Wi/interval	μm/d	Parfitt et al. (1987)
LGR	G-Int.Wi/interval	μm/d	Parfitt et al. (1987)
BFR/BS	(dL.Pm+sL.Pm/2) × MAR/Tb.Pm × 100	μm/d*100	Parfitt et al. (1987)
BFR/BV	(dL.Pm+sL.Pm/2) × MAR/1000/Tb.Ar × 365 × 100	%/year	Parfitt et al. (1987)
BFR/TV	(dL.Pm+sL.Pm/2) × MAR/1000/T.Ar × 365 × 100	%/year	Parfitt et al. (1987)

Tb.Ar, trabecular area; Tb.Th, trabecular thickness; Tb.N, trabecular number; Tb.Sp, trabecular separation; Tb.Pm, trabecular perimeter; T.Ar, tissue area; sL.Pm, single-labeled perimeter, dL.Pm, double-labeled perimeter; Int.Wi, interlabel width; G-Int.Wi, interlabel width in growth plate; %L.Pm, percent labeled perimeter; LGR, longitudinal growth rate; MAR, mineral apposition rate; BFR, bone formation rate; bone formation rate per unit of bone surface (BFR/BS), per unit of bone volume (BFR/BV) and per unit of tissue volume (BFR/TV).

Figure 1. (a) Representative fluorescence micrographs of G-Int.Wi on PTM (undecalcified sections, 8 μm, 10×). 1M: 1 month old, 3M: 3 months old, 6M: 6 months old, 14M: 14 months old. Arrows point to interlabel width after fluorochrome labeling with tetracycline hydrochloride and calcein. The interlabel width in the growth plate for 1-month-old rats was obviously wider than that for other groups, and it declined progressively with increasing age and was narrowest for 14-month-olds. The interlabel width in the growth plate was used to determine LGR. Quantitative measurements of LGR are shown in Figure 1b. (b) Changes of LGR in different ages rats. Data are expressed as means ± SD (n = 6) in each group. LGR was significantly increased in 1- and 3-month-old rats vs. 6- and 14-month-old rats, respectively, and with no differences for 6-month-old rats versus 14-month-old rats. Vs. 14-month-old,^*P < 0.01; vs. 6-month-old, ^▵P < 0.01, Student’s t-test. PTM, the proximal tibial metaphyses; LGR, longitudinal growth rate; G-Int.Wi, interlabel width in growth plate; 1M: 1-month-old, 3M: 3-month-old, 6M: 6-month-old, 14M: 14-month-old.

Figure 2. (a) Representative single-fluorescence micrographs of PTM in different ages rats (undecalcified sections, 8 μm, 20×). 1M: 1 month old, 3M: 3 months old, 6M: 6 months old, 14M: 14 months old. The clear and abundant single fluorescence could be seen in 1- and 3-month-old rats. There was less single fluorescence for 6-month-old rats and the minimum for 14-month-old rats, compared with 1- and 3-month-old rats. Arrows point to single fluorescence after fluorochrome labeling with tetracycline hydrochloride and calcein. Quantitative measurements of single fluorescence are shown in Figure 2b. (b) Changes of %L.Pm in rats of different ages. Data are expressed as means ± SD (n = 6) in each group. There were no significant differences in %L.Pm for 1-month-old rats vs. 6-month-old rats; however, this was significantly increased vs. 14-month-old rats, and significantly increased for 3-month-old vs. 6- and 14-month-old rats, respectively. Furthermore, there were no differences in %L.Pm for 6-month-old rats vs. 14-month-old rats. Vs. 14-month-old, ^◊P < 0.05, ^◊◊P < 0.01; vs. 6-month-old, ^#P < 0.05, Student’s t-test. PTM, the proximal tibial metaphyses; %L.Pm, percent labeled peri meter; 1M: 1-month-old, 3M: 3-month-old, 6M: 6-month-old, 14M: 14-month-old.

Figure 3. (a) Representative double-fluorescence micrographs of PTM in rats of different ages (undecalcified sections, 8 μm, 20×). 1M: 1 month old, 3M: 3 months old, 6M: 6 months old, 14M: 14 months old. A wider and longer double-fluorescence interval as well as an obvious yellow and green fluorescent strip could be seen in both 1- and 3-month-old rats, and these were narrower and shorter for 6-month-old rats and narrowest and shortest for 14-month-old rats. Arrows point to double fluorescence after fluorochrome labeling with tetracycline hydrochloride and calcein. Quantitative measurements of double fluorescence are shown in Figure 3b. (b) Changes of MAR in rats of different ages. Data are expressed as means ± SD (n = 6) in each group. MAR was significantly increased in 1- and 3-month-old rats vs. 6- and 14-month-old rats, respectively, and there were no differences for 6-month-old rats vs. 14-month-old rats. Vs. 14-month-old, ^*P < 0.01; vs. 6-month-old, ^▵P < 0.01, Student’s t-test. PTM, the proximal tibial metaphyses; MAR, mineral apposition rate; 1M: 1-month-old, 3M: 3-month-old, 6M: 6-month-old, 14M: 14-month-old.

Figure 4. Changes in bone formation, including BFR/BS, BFR/BV, BFR/TV, on PTM in rats of different ages. 1M: 1 month old, 3M: 3 months old, 6M: 6 months old, 14M: 14 months old. (a) Changes of BFR/BS on PTM. (b) Changes of BFR/BV on PTM. (c) Changes of BFR/TV on PTM. BFR/BS and BFR/BV, which peaked for 3-month-old rats, were significantly increased in 1- and 3-month-old rats compared with 6- and 14-month-old rats, respectively. Also, BFR/TV were significantly increased for 1- and 3-month-old rats compared to 14-month-old rats, whereas there were no differences compared to 6-month-old rats, respectively. Data are expressed as means ± SD (n = 6) in each group. Vs. 14-month-old, ^◊P < 0.05, ^◊◊ P < 0.01; vs. 6-month-old, ^#P < 0.05, ^##P < 0.01, Student’s t-test. PTM, the proximal tibial metaphyses; bone formation rate per unit of bone surface (BFR/BS), per unit of bone volume (BFR/BV) and per unit of tissue volume (BFR/TV). 1M: 1-month-old, 3M: 3-month-old, 6M: 6-month-old, 14M: 14-month-old.

Figure 5. (a) Effects of bone structure and trabecular mass on PTM in rats of different ages. 1M: 1 month old, 3M: 3 months old, 6M: 6 months old, 14M: 14 months old. Slender and interlacing trabeculae were found in 1- and 3-month-old rats. Cancellous bone mass was increased and trabeculae were thickened in 6-month-old rats compared with 1- and 3-month-old rats. Meanwhile, slight increases in cancellous bone mass were seen for 14-month-old rats compared to 6-month-old rats (silver nitrate stain, 4×; trabecular in black stain). Quantitative measurements of static parameters are shown in Figure 5b. (b) Static parameters of %Tb.Ar, Tb.Th, Tb.N and Tb.Sp, on the proximal tibia in rats of different ages. Data are expressed as means ± SD (n = 6) in each group. %Tb.Ar, percent trabecular area; Tb.Th, trabecular thickness; Tb.N, trabecular number; Tb.Sp, trabecular separation. %Tb.Ar, Tb.Th and Tb.N increased progressively, whereas Tb.Sp reduced gradually with aging. Vs. 14-month-old, ^◊◊ P < 0.01; vs. 6-month-old, ^##P < 0.01, Student’s t-test. PTM, the proximal tibial metaphyses; Tb.Ar, trabecular area; Tb.Th, trabecular thickness; Tb.N, trabecular number; Tb.Sp, trabecular separation. 1M: 1-month-old, 3M: 3-month-old, 6M: 6-month-old, 14M: 14-month-old.

Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ, Ott SM, Recker RR. 1987. Bone histomorphometry: Standardization of nomenclature, symbols, and units: Report of the ASBMR histomorphometry nomenclature committee. Journal of Bone and Mineral Research 2:595–610. doi:10.1002/jbmr.5650020617.

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