A quantitative analysis of bone lamellarity and bone collagen linearity induced by distinct dosing and frequencies of teriparatide administration in ovariectomized rats and monkeys

Microscopy, 2021, 498–509 doi:https://doi.org/10.1093/jmicro/dfab020 Advance Access Publication Date: 8 June 2021 Article Takanori Sato1 , Aya Takakura1,2,* , Ji-Won Lee1 , Kazuaki Tokunaga3 , Haruka Matsumori3 , Ryoko Takao-Kawabata2,* and Tadahiro Iimura1,* 1 Department of Pharmacology, Faculty and Graduate School of Dental Medicine, Hokkaido University, N13 W7, Sapporo, Hokkaido 060-8586, Japan; 2 Pharmaceuticals Research Center, Asahi Kasei Pharma Corporation, 632-1 Mifuku, Izunokuni, Shizuoka 410-2321, Japan and 3 Nikon Corporation, 2-15-3 Konan, Minato-ku, Tokyo 108-6290, Japan *To whom correspondence should be addressed. E-mail: (A.T.); (R.T.); (T.I.) Received 16 March 2021; Revised 19 May 2021; Editorial Decision 5 June 2021; Accepted 7 June 2021 Abstract The lamellar structure of bone, which endows biomechanical rigidity to support the host organism, is observed in mammals, including humans. It is therefore essential to develop a quantitative analysis to evaluate the lamellarity of bone, which would especially be useful for the pharmacological evaluation of anti-osteoporotic drugs. This study applied a current system for the semi-automatic recognition of fluorescence signals to the analysis of un-decalcified bone sections from rat and monkey specimens treated with teriparatide (TPTD). Our analyses on bone formation pattern and collagen topology indicated that TPTD augmented bone lamellarity and bone collagen linearity, which were possibly associated with the recovery of collagen cross-linking, thus endowing bone rigidity. Key words: bone lamellarity, collagen linearity, multi-photon microscopy, SHG (second harmonic generation), semi-automatic recognition, teriparatide Introduction The most characteristic feature of vertebrates is the bone, which is mainly composed of collagen fibers and hydroxyapatite. The evolution of bone has been associated with the development of a hormonal system that regulates mineral metabolism. The parathyroid hormone (PTH) plays key roles in the homeostasis of calcium phosphate through its actions in the bone and kidneys, and indirectly in the intestine [1–3]. Furthermore, the functional evolution of PTH has been thought to be a key event in appearance of terrestrial vertebrates [4–6]. In skeletal ontogeny, primary bone develops in a de novo manner, where no bone existed previously, in given parts of fetal mesenchymal tissue, which is structurally termed woven bone. Then, mature bone develops in a manner in which previously formed bone (either woven bone or lamellar bone) is replaced by new lamellar bone [7]. It is understood that woven bone forms when rapid bone formation is required, such as in early skeletal development and bone repair. Thus, woven bone appears to be formed as a ‘place-taking’ bone for future mature bone formation. Lamellar bone has a highly organized layered structure with well-oriented thick collagen fibers, whereas © The Author(s) 2021. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. 498 This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact A quantitative analysis of bone lamellarity and bone collagen linearity induced by distinct dosing and frequencies of teriparatide administration in ovariectomized rats and monkeys T. Sato et al. Quantitative analysis of bone lamellarity and bone collagen linearity Moreover, this method requires a high degree of skill for histological observation and thus shows a degree of subjectivity. This study applied a current semi-automatic recognition system to the nondemineralized histological bone sections with calcian labeling and Villanueva bone staining, which are well-established histomorphometric methods that are used to evaluate the rate of bone turnover. We also subjected these bone sections to collagen imaging by second harmonic generation (SHG) [20,23–27]. To statistically analyze the spatial pattern of bone formation associated with bone collagen, we re-evaluated previously reported rat and cynomolgus monkey bone specimens that were treated with TPTD [19,28,29]. Materials and methods Animals and experimental design All experimental protocols were approved by the experimental animal ethics committee of Asahi Kasei Pharma Co., and were conducted in accordance with guidelines concerning the management and handling of experimental animals. Rats: This study re-examined rat bone specimens that we previously analyzed and reported [19]. Briefly, sexually mature female Sprague-Dawley rats (Charles River, Kanagawa, Japan) of 13 weeks of age were divided into 15 drug regimen groups (Supplementary Fig. 1). Ovariectomy (OVX) or sham surgical operations were conducted under anesthesia, as previously described. At 16 weeks of age, the rats were subcutaneously injected with vehicle saline, or 6 or 30 µg/kg of TPTD acetate (Asahi Kasei Pharma Corporation, Tokyo, Japan), 3 times/week (W3 groups, marked in red); vehicle, 1.2, 6 or 30 µg/kg of TPTD once a day (D1 groups, in orange) or twice a day (D2 groups, in green); and vehicle, 1.2 or 6 µg/kg of TPTD 3 times/day (D3 groups, in blue) for 4 weeks. On the eighth and third days before sacrifice, double-fluorochrome labeling was conducted with two subcutaneous injections of calcein (Dojindo Laboratories, Kumamoto Japan) into each rat at a dose of 10 mg/kg body weight, in order to observe sites of active bone formation. Monkeys: This study also re-examined the bone specimens of female cynomolgus monkeys (C.V. Universal Fauna, Jakarta Timur, Indonesia), as previously analyzed and reported [28]. Monkeys (age: 12.0 ± 1.5 years, weight: 2.06–3.48 kg) were divided into four groups (n = 19–20): (i) the Sham group; (ii) OVX group; (iii) the TPTD-L group, OVX with low-dose TPTD (1.2 µg/kg, once a week); and (iv) the TPTD-H group, OVX with high-dose TPTD (6.0 µg/kg, once a week) (Supplementary Fig. 2). OVX and sham operations were carried out 1 week before TPTD treatment. TPTD or saline (as a vehicle) was subcutaneously injected once a week, for 18 months (Supplementary Fig. 2a). To determine bone formation rates and sites of active mineralization, double-fluorochrome labeling with intravenous calcein injection (4 mg/kg; Dojindo Laboratories, Kumamoto, Japan) at Days 7 and 21 before sacrifice was conducted for all monkeys. Bone histomorphometry and selection of specimens for the semi-automatic imaging analysis Bone histomorphometry was performed on the sagittal sections of second lumbar vertebra from all monkey specimens to evaluate bone mass, structure and bone metabolism (resorption and formation) in cancellous bone using an image analysis system (Histometry RT Camera, System Supply, Nagano, Japan; OsteoMeasure, Decatu (...truncated)