Mechanical Loading Stimulates Differentiation of Periodontal Osteoblasts in a Mouse Osteoinduction Model: Effect on Type I Collagen and Alkaline Phosphatase Genes D. Pavlin, 1 S. B. Dove, 3 R. Zadro, J. Gluhak-Heinrich 2 1 Departments of Orthodontics and Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio, Texas 78284-7910, USA 2 Department of Orthodontics, The University of Texas Health Science Center at San Antonio, Antonio, Texas 78284-7910, USA 3 Department of Dental Diagnostic Science, The University of Texas Health Science Center at San Antonio, Antonio, Texas 78284-7910, USA Received: 9 August 1999 / Accepted: 4 February 2000 Abstract. The effects of mechanical loading on the osteo- blast phenotype remain unclear because of many variables inherent to the current experimental models. This study re- ports on utilization of a mouse tooth movement model and a semiquantitative video image analysis of in situ hybrid- ization to determine the effect of mechanical loading on cell-specific expression of type I collagen (collagen I) and alkaline phosphatase (ALP) genes in periodontal osteo- blasts, using nonosseous cells as an internal standard. The histomorphometric analysis showed intense osteoid deposi- tion after 3 days of treatment, confirming the osteoinductive nature of the mechanical signal. The results of in situ hy- bridization showed that in control periodontal sites both collagen I and ALP mRNAs were expressed uniformly across the periodontium. Treatment for 24 hours enhanced the ALP mRNA level about twofold over controls and maintained that level of stimulation after 6 days. In contrast, collagen I mRNA level was not affected after 24 hours of treatment, but it was stimulated 2.8-fold at day 6. This in- crease reflected enhanced gene expression in individual os- teoblasts, since the increase in osteoblast number was small. These results indicate that (1) the mouse model and a semi- quantitative video image analysis are suitable for detecting osteoblast-specific gene regulation by mechanical loading; (2) osteogenic mechanical stress induces deposition of bone matrix primarily by stimulating differentiation of osteo- blasts, and, to a lesser extent, by an increase in number of these cells; (3) ALP is an early marker of mechanically- induced differentiation of osteoblasts. (4) osteogenic me- chanical stimulation in vivo produces a cell-specific 2.8-fold increase in collagen gene expression in mature, matrix- depositing osteoblasts located on the bone surface and within the osteoid layer. Key words: Osteoblast differentiation — Gene regulation by mechanical stress — Alkaline phosphatase — Type I collagen — Mouse periodontium. Mechanical stress is an important regulatory factor in bone homeostasis and a determinant of skeletal morphology from early developmental stages and throughout the lifetime. Be- cause of its influence on and interactions with all other regulators of bone metabolism, the effect of mechanical loading on proliferation and phenotype expression in osteo- blasts has been extensively studied in the past. Two of the most common parameters for determining the mechanical effect on osteoblasts are the expression of type I collagen (collagen I) and alkaline phosphatase (ALP), molecules as- sociated with the osteoblast phenotype. Several cell culture models have been used in the past to determine changes in these osteoblast markers under various types of mechanical stress. Intermittent hydrostatic compression (IHC) enhanced ALP enzyme activity in monolayer cultures of MC3T3 os- teoblastic cells [1], fetal mouse calvaria organ cultures [2], and ROS17/2.8 cells [3], whereas a continuous hydrostatic compression suppressed this enzyme activity in MC3T3 cells [4]. Further studies suggested the role of IHC in main- taining a mature osteoblast phenotype, since its application restored the loss of ALP gene expression and enzyme ac- tivity observed in control cultures in the absence of me- chanical stimulation [5]. In contrast to the effect on ALP, intermittent hydrostatic pressure did not affect the level of collagen I mRNA in ROS17/2.8 cells [3], but it enhanced the collagen I protein synthesis in primary calvarial osteo- blasts [1, 6], and maintained the collagen I mRNA level in collagen-declining calvarial organ cultures [5]. A continu- ous compressive force, however, inhibited collagen I in MC3T3 cells [4]. Deformation of cells by stretching on flexible mem- branes produced variable results in the effect on ALP and collagen I. The ALP enzyme activity decreased in primary calvarial osteoblast cultures and MC3T3 cell line exposed to a low level strain considered to be within the physiological range [7, 8], but only after a prolonged period of treatment for 10 days [9]. Similar inhibitory response of ALP was observed in an earlier study on calvarial osteoblastic cells under low strain, but the response was noted after 24 hours of stretching [10]. In contrast, the application of high strain (24%) resulted in a twofold increase of ALP activity in UMR cells after 24 hours [11], and a 1% strain applied to primary digest from human femur had no effect on this enzyme [12]. Reduced atmospheric pressure during the space flight (microgravity) had a twofold stimulatory effect on ALP in the ROS17/2.8 cell line after 6 days [13]. In Present address: Department of Laboratory Medicine, Clinical Hospital Center, The University of Zagreb, Croatia Correspondence to: D. Pavlin, Department of Orthodontics Calcif Tissue Int (2000) 67:163–172 DOI: 10.1007/s00223001105 © 2000 Springer-Verlag New York Inc.