Endochondral ossification in vitro is influenced by mechanical bending

Bone 40 (2007) 597 – 603 www.elsevier.com/locate/bone

Endochondral ossification in vitro is influenced by mechanical bending☆ Britta Trepczik a,1 , Jasmin Lienau a,1 , Hanna Schell a , Devakara R. Epari a , Mark S. Thompson a , Jan-Erik Hoffmann a , Anke Kadow-Romacker a , Stefan Mundlos b , Georg N. Duda a,⁎ a

Center for Musculoskeletal Surgery, Charité – Universitätsmedizin Berlin, Augustenburger Platz 1, D-13353, Berlin, Germany b Institute for Medical Genetics, Charité – Universitätsmedizin Berlin, Germany Received 8 May 2006; revised 31 August 2006; accepted 11 October 2006 Available online 30 November 2006

Abstract Bone development is influenced by the local mechanical environment. Experimental evidence suggests that altered loading can change cell proliferation and differentiation in chondro- and osteogenesis during endochondral ossification. This study investigated the effects of three-point bending of murine fetal metatarsal bone anlagen in vitro on cartilage differentiation, matrix mineralization and bone collar formation. This is of special interest because endochondral ossification is also an important process in bone healing and regeneration. Metatarsal preparations of 15 mouse fetuses stage 17.5 dpc were dissected en bloc and cultured for 7 days. After 3 days in culture to allow adherence they were stimulated 4 days for 20 min twice daily by a controlled bending of approximately 1000–1500 microstrain at 1 Hz. The paraffin-embedded bone sections were analyzed using histological and histomorphometrical techniques. The stimulated group showed an elongated periosteal bone collar while the total bone length was not different from controls. The region of interest (ROI), comprising the two hypertrophic zones and the intermediate calcifying diaphyseal zone, was greater in the stimulated group. The mineralized fraction of the ROI was smaller in the stimulated group, while the absolute amount of mineralized area was not different. These results demonstrate that a new device developed to apply three-point bending to a mouse metatarsal bone culture model caused an elongation of the periosteal bone collar, but did not lead to a modification in cartilage differentiation and matrix mineralization. The results corroborate the influence of biophysical stimulation during endochondral bone development in vitro. Further experiments with an altered loading regime may lead to more pronounced effects on the process of endochondral ossification and may provide further insights into the underlying mechanisms of mechanoregulation which also play a role in bone regeneration. © 2006 Elsevier Inc. All rights reserved. Keywords: Bone development; Endochondral ossification; Histomorphometry; Mechanical stimulation; Organ culture

Introduction The mechanical environment is important for the proliferation and differentiation of cells and tissues. An altered loading regime during the development of long bones can lead to mechanically induced changes in the process of endochondral ossification. The development of the growing bone anlagen is influenced by the muscular-induced loading in the prenatal stage. The inhibition of muscular activity in human fetuses ☆ No benefit of any kind has been or will be received either directly or indirectly by the authors. ⁎ Corresponding author. Fax: +49 30 450 559969. E-mail address: [email protected] (G.N. Duda). 1 Shared first authorship.

8756-3282/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2006.10.011

decreased osteogenesis resulting in bone with reduced diameter and cortical thickness. These bones were prone to fractures and showed signs of osteoporosis [1]. Distortional strains, resulting from muscle contractions, were shown to modulate the shape of the mineralization front of the primary ossification center in murine metatarsal bones in vivo. The lack of this loading led to a slower mineralization rate at the periphery of the bones that had developed in vitro compared to bones at the same stage that had developed in utero [2]. The effect of various forms of biophysical stimulation on the process of endochondral ossification has been the subject of numerous studies. Dynamic cell stretching has been shown to stimulate osteoblast proliferation in vitro [3]. Cyclic strain at a physiologic magnitude of 1000 microstrain (μE) led to an

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increase of osteoblasts activities related to matrix production while those activities relevant for matrix mineralization were decreased [4]. In vitro organ culture models of fetal murine metatarsal bones have been used to determine the effects of different types of biophysical stimulation on the process of endochondral ossification. The development of these long bones involves different cellular processes including chondrocyte proliferation and hypertrophy as well as matrix mineralization and bone collar formation which may be differentially influenced by the applied stimulation. Hydrostatic compression was found to stimulate chrondrocyte hypertrophy and extracellular matrix mineralization [5–7]. The hydrostatic pressure produced significant shear stresses at mineralized/non-mineralized tissue interfaces influencing matrix-producing (chondrocytes, osteoblasts) and matrix-resorbing cells (osteoclasts) in vitro [8,9]. Stimulation with low intensity pulsed ultrasound in the murine bone culture model was shown to increase bone cell differentiation, leading to an elongation of the bone collar, and to an increase in the calcified matrix production of developing bones [10,11]. The longitudinal bone growth, which is mainly caused by chondrocyte proliferation, was not changed by stimulation in any of the aforementioned studies. The mechanical environment is also known to be important for the maintenance and regeneration of the adult skeletal system. Endochondral ossification during bone healing has been described as a recapitulation of the processes seen during fetal development of the long bones [12,13]. Therefore, the organ culture model of fetal long bone rudiments is highly suitable to analyze the influence of mechanical stimulation on endochondral ossification, a particular aspect of bone regeneration. The aim of this study was to investigate the influence of a controlled loading regime on cartilage differentiation and calcification as well as bone collar formation in the early development of long bones. In contrast to other forms of stimulation e.g. air pressure or ultrasound [5,7,9–11], the loads applied in this study were intended to mimic the native loads developed by the plantar muscles on the metatarsal bones. For this purpose a new testing device was constructed to apply mechanical stimulation in the form of cyclic bending to murine fetal metatarsal bones in vitro. The effects of the loading were determined by histomorphometrical analysis. In accordance with previous studies [7,10,11], we hypothesized that mechanical loading does not change length or width of the whole bone anlagen, but rather leads to an enhancement of bone formation resulting in an elongated periosteal bone collar and an increased calcification in the diaphysis. As calcification of cartilage is preceded by hypertrophy of the cartilaginous tissue, we also expected an enhancement in the size of the hypertrophic zones. Materials and methods Mice The experiments were approved by the local legal representative (LAGetSi; Reg.-No. T 0159/03). Four 10- to 16-week-old female C57Bl/6 wild-type mice (Harlan-Winkelmann, Borchen, Germany) were used. Timed matings were

produced and confirmed with identification of the vaginal plug. Noon of this day was defined as 0.5 days post coitus (dpc). The gestating mice were sacrificed at 17.5 dpc and the metatarsal bones (MT) of 15 mice fetuses were dissected.

Experimental procedure and tissue culture Each foot was separated from the lower leg proximally from the ankle under sterile conditions in phosphate-buffered saline (1× PBS). Skin, toes and tarsal bones were carefully removed without disrupting the intermediate tissue. The cartilaginous anlagen of MT II to V of the fetuses were placed en bloc with the plantar side on a round nitrocellulose filter with 14 mm diameter, 180 μm thickness and 0.8 μm pore size (AA BP 02500, Millipore, Germany). The filter with the tissue on top was placed on a dentin chip with 14 mm diameter and 0.7 mm thickness. The chip was then placed in the mechanical testing device (Fig. 1) fitting in a well of the 12-well culture plate. The specimens were cultured in the mechanical device during the entire time and each placed separately in one well of the 12-well plate. From the two specimens (left and right metatarsus) of each fetus, one was stimulated while the other served as control. The stimulated bones and the corresponding controls were cultured in the same culture plate. The MT were completely covered with standardized fluid culture medium consisting of alpha minimal essential medium with nucleosides (αMEM F0915; Biochrom AG, Berlin, Germany), supplemented with 0.1% bovine serum albumin (BSA; Fluka 05488, Sigma, Germany), 0.6 mM L-ascorbic acid (A89605G; Sigma, Germany), 1.37 mM L-glutamine (1680146; ICN Biomedicals, USA), 1 mM β-glycerophosphate-Mg (G-9891; Sigma, Germany) and 1× antibiotic/antimycotic solution (Gibco-BRL No. 15240-096, Life Technologies, USA). The culture plates were kept in a humidified 5% CO2 incubator at 37°C for 7 days except for the stimulation time. The medium was not renewed during the experiment to allow biochemical interaction of the cells by secreted mediators. The controls were kept under identical conditions but lacked stimulation.

Mechanical stimulation The metatarsal preparations were given three days to adhere to the filters. After 72 h, they were stimulated mechanically 20 min every 12 h for a period of 4 days. Stimulation was achieved by a three-point bending of the dentin chip together with filter and adherent tissue. The specimens were positioned

Fig. 1. Schematic of mechanical testing device. The device consists of the upper part (1) with two side parts (2) of different length (represented by broken lines) for stimulated and control devices; the specimen (3) is placed on the filter (thick line) on top of the dentin chip (4); the fundamental structure bears the bending tip (5) and two flexible plastic tubes (6), where the chip is resting if not stimulated. Loading of upper part (1) results in three-point bending of the elastic dentin chip (4). The arrows represent the loading direction; not shown is the outer guidance of the device.

B. Trepczik et al. / Bone 40 (2007) 597–603 in a way such that the bending was along the long axis of the bones at a frequency of 1 Hz. The loading was applied under displacement control until a maximum displacement of 0.6 mm was reached. The displacement to be applied to the three-point bending device was determined in preliminary tests. Using an Instron materials testing machine (8871, Instron, Germany) to apply the loads and a strain gauge to measure the strains on the surface of the dentin chip, it was determined that a displacement of 0.6 mm was necessary to produce strains of the order of 1000–1500 μE. To allow for possible softening of the dentin chip due to its immersion in the culture medium during the experiments, this analysis was performed after soaking the dentin chip in medium for 24 h. To confirm that the straining of the dentin chip was transferred to the metatarsal anlagen high-resolution photographs during bending were taken. The photographs demonstrated conformity between the dentin chip and the bone anlagen throughout a complete loading and unloading cycle (Fig. 2). Furthermore, these images also enabled the change in length of the anlagen themselves to be estimated using Digital Image Correlation (DIC: Vic2D, Correlated Solutions, West Columbia, USA), confirming gross strain magnitudes of the order of 1000 μE. Stimulation was performed at room temperature in the closed culture plates by pressing down the devices along with the lid of the plate. Loading was carried out with an Instron Materials' testing machine. The device was designed in two slightly different types to allow stimulated and control specimens of each fetus in the same culture plate (see Fig. 1).

Histology After 7 days in culture the tissue blocks were fixed in 4% phosphatebuffered paraformaldehyde (PFA/PBS) overnight at 4°C. After washing twice in PBS, they were dehydrated through a graded ethanol series and embedded in paraffin. Serial sections of 5 μm were cut parallel to the plantar side of the foot with a microtome (RM 2125, Leica, Germany). This resulted in 12 to 18 microscope slides with 2 sections on each. All steps were carried out with the metatarsal preparations still adherent on the subjacent filters. Every third slide (1st, 4th, 7th, …) was stained with hematoxylin/eosin (HE) for general examination of morphology and vitality of the tissue. Safranin Orange/von Kossa (SO/vK) staining [14] was similarly performed on every other third slide (2nd, 5th, 8th, …) for histomorphometrical analysis.

Histomorphometrical analysis Computerized histomorphometrical analysis of SO/vK stained metatarsal bones II to IV was performed with an image analysis system (KS400, Zeiss, Germany). For each MT, the section analyzed corresponded to the section that was cut most closely in the central plane of the bone. Therefore, the three different analyzed bones of each preparation were not necessarily obtained from the same microscopical section. The ROI comprised the two hypertrophic zones and the intermediate diaphyseal calcifying zone. The bone ends containing the zones of resting and proliferating chondrocytes, the periosteum and the periosteal bone collar were excluded from the ROI. In addition, the length of the whole bones and the medial and lateral periosteal bone collars were measured. The lengths, widths and areas of the hypertrophic zones and the zone of mineralization were quantified. The length of each zone was determined along the centerline of each

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bone. Due to the hourglass shape of the developing MT, the maximum widths of hypertrophic zones and minimum width of calcifying zone were taken. The mineralized area was determined from segmentation of the black regions of the SO/vK stain. The length of the ROI relative to the total bone length and the fraction of mineralized area of ROI were calculated from the data.

Statistical analysis To determine statistical differences between stimulated and control groups, a three-factorial analysis of variance for repeated measures according to Brunner and Puri [15] was performed using SAS (SAS V8, SAS Institute Inc., Cary, NC, USA). This kind of analysis was chosen because normal distribution of the data could not be assumed. Distributions of the measured parameters were characterized by median and 25–75 percentiles (Table 1). The tested hypothesis was always that stimulation does not alter the parameter. A p-value of less than 0.05 was considered significant.

Results Histology The Hematoxylin/Eosin stained sections of the metatarsal preparations demonstrated a healthy normal appearance in all specimens. The general histological outcome revealed no differences in morphology and vitality of the tissue between stimulated and control groups. All bone anlagen showed the different zones of resting, proliferating and hypertrophic cartilage with the calcifying diaphyseal zone in the center. The calcifying zone was flanked by the periosteal bone collar, which extended along the distal and proximal hypertrophic zones (Fig. 3). Histomorphometry The histomorphometrical analysis (Table 1) revealed no differences in the length of the whole metatarsal bones (MT) and the ROI between loaded and control groups. The length of the ROI itself, consisting of the intermediate calcifying and the two hypertrophic zones, constituted about half the length of the whole bones. The periosteal bone collar was slightly shorter than the whole ROI (Table 1). The collar on the lateral side was significantly longer in the stimulated group (median [25–75 percentile]: 735 [655–819] μm) compared to controls (731 [636–792] μm; p = 0.039). The medial periosteal bone collar of the stimulated bones tended to be longer than controls (769 [705–815] μm to 750 [666–807] μm; p = 0.172). Considering the individual

Fig. 2. High-resolution photographs of a metatarsal bone preparation during bending demonstrating conformity between the dentin chip and the bone anlagen throughout a complete loading cycle (left: null position, right: maximum displacement). A dotted line has been added to indicate the upper surface of the dentin chip.

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Table 1 Distribution characteristics of murine fetal metatarsal bones (MT) Parameter

Length of MT

Length of ROI

Mineralized area

Width of proximal hypertrophic zone

Width of distal hypertrophic zone

Total hypertrophic zone

Area of calcified zone

Mineralized area of proximal hypertrophic zone

Mineralized area of distal hypertrophic zone

Mineralized area of calcified hypertrophic zone

MT

II III IV II III IV II III IV II III IV II III IV II III IV II III IV II III IV II III IV II III IV

Control

Stimulated

Median

25–75 percentiles

Median

25–75 percentiles

1375 1451 1510 770 789 804 35.7 36.0 42.7 382 367 372 449 435 457 135.2 130.5 136.2 124.0 126.5 152.3 5.4 4.9 7.1 5.1 5.4 6.1 24.5 23.3 29.4

[1313–1450] [1401–1514] [1438–1557] [704–808] [724–806] [724–844] [29.6–51.5] [29.0–45.5] [37.2–55.5] [352–391] [356–385] [359–381] [423–468] [422–453] [446–482] [107.9–159.3] [112.1–166.2] [114.0–166.3] [109.5–145.8] [104.4–150.3] [121.0–184.6] [3.0–8.4] [2.9–6.8] [5.0–7.8] [3.1–11.6] [3.6–9.3] [3.1–9.4] [21.8–32.2] [22.0–32.0] [23.4–36.8]

1399 1472 1518 774 771 810 35.9 35.6 41.4 376 365 374 457 435 472 135.4 129.3 132.5 135.5 129.2 147.2 4.2 4.7 5.5 6.4 4.8 5.7 25.9 24.3 28.5

[1339–1429] [1421–1542] [1435–1584] [706–818] [724–855] [784–871] [26.0–45.8] [28.5–44.9] [32.3–54.5] [335–397] [357–371] [362–390] [430–469] [433–452] [456–483] [123.0–147.4] [105.6–150.8] [123.6–155.4] [104.1–154.1] [116.4–150.5] [135.0–185.5] [1.7–6.7] [2.5–6.6] [1.5–6.4] [2.5–7.4] [3.6–6.9] [5.1–10.4] [16.0–32.9] [21.0–33.2] [21.4–39.3]

pvalue 0.332

0.231

0.700

0.994

0.070

0.666

0.137

0.072

0.249

0.635

Median values [25–75 percentiles]; the three MT are shown separately (n = 15 each/group); the p-value was calculated for the whole distribution of stimulated vs. control group (n = 45 MT). Units of measurements are [μm] for distances and [× 103 μm2] for areas.

bones MT II to IV, the lateral and medial bone collars of the stimulated MT III and IV were longer compared to the controls, while the bone collar of MT II was of equal size (Fig. 4). Mechanically loaded bones had a significantly greater ROI size than the controls (286.9 [252.8–313.4] × 103 μm2 to 272.3 [253.0–289.3] × 103 μm2; p = 0.033). Of the three individual bones, MT IV showed the greatest increase in ROI size, the size of the ROI of MT II was enhanced as well, while for MT III stimulated and control specimen were of equal size (Fig. 5). The absolute amount of mineralized tissue in the stimulated group was not different from controls (Table 1). However, the fraction of mineralized tissue in the ROI was significantly lower for the loaded specimen (14.0% [10.8–16.1%] to 14.9% [11.5– 18.3%]; p = 0.031). Examination of the individual metatarsals revealed a diminished percentage of calcification for loaded MT II and IV, while there was no difference in MT III between both groups (Fig. 6). The lengths and widths of the three distinct zones of the ROI were not significantly different in stimulated and control bones. For both groups the proximal hypertrophic zone was slightly longer than the distal one. Both hypertrophic zones together had nearly the same length as the intermediate calcified zone. The

distal hypertrophic zone was about 20% wider compared to the proximal one (Table 1). The distal hypertrophic zone of the stimulated group tended to be wider than the controls' (p = 0.070). The areas of the three parts of the ROI revealed no significant differences between loaded bones and controls. The size of the mineralized area was not changed by the mechanical stimulation in the calcified diaphyseal and distal hypertrophic zone (Table 1). In the proximal hypertrophic zone the amount of mineralized area tended to be lower in the stimulated bones (p = 0.072). Discussion In this study, a newly developed testing device was used to apply a controlled bending load and the effect of this mechanical stimulation on cartilage differentiation and calcification as well as bone collar formation in the early development of murine fetal metatarsal bone anlagen cultured in vitro was analyzed. In contrast to other forms of stimulation e.g. air pressure or ultrasound [5,7,9–11], the loading applied in this study was

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Fig. 3. Histological section of SO/vK-stained MT preparation after 7 days of culture. Above: MT III with histomorphometrically marked ROI with calcified (dark green) and hypertrophic zones (red) and mineralized tissue (light green). Below: same MT with marked proximal/distal hypertrophic zones (phyp/dhyp), calcified diaphyseal zone (calz), periosteal bone collar (arrows, poB). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

intended to mimic the native loading applied to the metatarsal bones by the developing plantar muscles. To our knowledge, this is the first study reporting the effects of this type of mechanical stimulation on metatarsal development. Endochondral bone formation during the development of long bones involves different cellular processes including chondrocyte proliferation and hypertrophy as well as matrix mineralization and bone collar formation. In this study, enhanced bone formation was seen as an elongation of the bone collar in the stimulated samples. Other studies applying various forms of biophysical stimulation in the metatarsal bone (MT) culture system reported diverse results. Van't Veen et al.

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Fig. 5. Size of ROI. The three MT are shown separately (n = 15 each); p-value for whole distribution of stimulated vs. control group (n = 45 MT; p = 0.033).

[7] found that the application of intermittent hydrostatic compressive force (ICF) for 5 consecutive days did not result in a lengthening of the bone collar. In contrast, Nolte et al. [10] described a slightly more pronounced bone collar around the central diaphysis following stimulation with low intensity pulsed ultrasound for 20 min/day over 7 days. Using the same model as Nolte but with treatment times of 3 and 6 days, Korstjens et al. [11] observed a significant increase in bone collar volume but not in length. These conflicting results do not suggest a general correlation between biophysical stimulation and bone collar length. In this study, differences were observed between the lateral and medial bone collars. These differences may be related to the

Fig. 4. Length of medial and lateral periosteal bone collar. The three MT are shown separately (n = 15 each); p-value was determined for whole distribution of stimulated vs. control group (n = 45 MT; p = 0.172 medial, p = 0.039 lateral).

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Fig. 6. Mineralized fraction of ROI. The three MT are shown separately (n = 15 each); p-value for whole distribution of stimulated vs. control group (n = 45 MT; p = 0.031).

sample preparation of the metatarsal bones. The bone collars of MT III and IV were situated inside the tissue block, while the medial bone collar of MT II was situated on the outer border. The lateral bone collars of all three analyzed bones were situated inside the tissue block so they were better protected during preparation and subjected to more physiologic like conditions during the entire culture time. Therefore, the significant enhancement of the lateral bone collar maybe related to more favorable conditions inside the tissue block which probably better represent the conditions in vivo. In addition to the enhanced bone collar formation, we found a significantly greater size of the ROI, consisting of the intermediate diaphyseal and the two hypertrophic zones, in the stimulated group compared to controls. However, the larger ROI could not be attributed to an increase in any particular ROI zone. Neither the length of the whole bones, nor the length or width of the individual zones was different in the loaded bones compared to the controls. But, the distal hypertrophic zone of the stimulated group did tend to be wider than that of the controls, which may partly explain for the greater ROI area determined. The three metatarsals contributed in varying degrees to the greater ROI area, with the greatest difference in ROI area occurring in the fourth metatarsal. Van't Veen et al. [7] measured in their ICF-stimulation study the total length of the three zones and the length of the intermediate mineralized zone separately and determined an increase in the length of the inner calcified zone. However, the metatarsal bones were not different in their total length or in the length of their hypertrophic cartilage zones, including the intermediate calcified zone [7]. Nolte et al. [10] determined a three-fold greater increase in the length of the inner calcifying zone compared to controls after ultrasound stimulation. The calcified matrix of the hypertrophic cartilage zone seemed more pronounced in the ultrasound stimulated bones than in the controls, but this was

not quantitatively determined. Korstjens et al. [11] found an increase in the percentage of calcified cartilage and in calcified cartilage volume for the ultrasound stimulated bone anlagen. The length of the calcified zone was in contrast to the results of Nolte et al. [10] not altered by ultrasound stimulation. Both these studies found no differences in total bone measures and lengths and widths of the individual zones. In accordance with the above cited studies [7,10,11], this study also demonstrated no change in the total bone length and the lengths and widths of the distinct zones due to the mechanical stimulation suggesting that mechanical stimulation did not affect proliferating chondrocytes. However, in apparent contradiction, no enhancement in the amount of matrix calcification could be determined as a result of mechanical stimulation. The total amount of mineralized area was not different for the mechanically stimulated group of metatarsal preparations in our experiments suggesting that mechanical stimulation did also not affect cellular processes involved in chondrocyte hypertrophy and matrix mineralization. In contrast, in the stimulated bones, the calculated mineralized fraction of the ROI was significantly smaller, due to the greater size of the ROI. The present study design with this new stimulation device was subject to several limitations. As the preparations were adhered to the nitrocellulose filters, stimulation of the bones was reliant on this bond not failing. In establishing the system, the strength of that adhesion was tested with several MT preparations by taking a sequence of high-resolution images during stimulation. From the inspection of these images, the metatarsal bone preparations seemed to be attached to the underlying filter (Fig. 2). A strong bond between the filter and the dentin chip was ensured by the design of the testing device (Fig. 1). In addition, the filter itself influences the growth of the bones in terms of decelerating the growth and the bones do not evolve in the same way as preparations floating free in culture medium (data not shown). However, in this study stimulated and control specimens would be influenced in a similar manner. The movement of the fluid medium in the culture dishes should also be considered [16]. During the performed stimulation the mechanical device was pressed down inside the well in a frequency of 1 Hz leading to a visible fluid flow of the medium only for the stimulated specimen. This may result in an enhanced exchange between culture medium and organ culture. Fluid flow is discussed to be one possible mediator of mechanotransduction [17,18]. In comparison to other studies where MT anlagen were stimulated constantly for 5 days by intermittent compressive force [5,7] or up to 7 days repeatedly by ultrasound stimulation [10,11], the loading in the present study was only performed for 4 days which might not be sufficient to yield substantial effects on the process of endochondral ossification. Whether the culture duration of 3 days prior to stimulation is really necessary for adequate adherence will be tested in future experiments. In summary the present study demonstrated that a newly developed device allowing a controlled bending caused an enhanced bone formation, resulting in a longer bone collar, but did not lead to a modification in cartilage differentiation and

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matrix mineralization. The results corroborate the influence of biophysical stimulation during endochondral bone development in vitro. Further experiments with an altered loading regime may lead to more substantial effects on matrix calcification or other processes of endochondral bone formation. Understanding of the involved developmental processes may provide further insight into the underlying mechanisms of mechanoregulation which also play a role in bone regeneration. From a clinical point of view, a better understanding of the influence of the mechanical conditions on bone modeling and remodeling may help to enhance the treatment of bone diseases or bone healing processes. Acknowledgments This study was supported by a grant of the Collaborative AO Research Center Berlin. References [1] Rodriguez JI, Palacios J, Garcia-Alix A, Pastor I, Paniagua R. Effects of immobilization on fetal bone development. A morphometric study in newborns with congenital neuromuscular diseases with intrauterine onset. Calcif Tissue Int 1988;43:335–9. [2] Tanck E, Blankevoort L, Haaijman A, Burger EH, Huiskes R. Influence of muscular activity on local mineralization patterns in metatarsals of the embryonic mouse. J Orthop Res 2000;18:613–9. [3] Neidlinger-Wilke C, Wilke HJ, Claes L. Cyclic stretching of human osteoblasts affects proliferation and metabolism: a new experimental method and its application. J Orthop Res 1994;12:70–8. [4] Kaspar D, Seidl W, Neidlinger-Wilke C, Ignatius A, Claes L. Dynamic cell stretching increases human osteoblast proliferation and CICP synthesis but decreases osteocalcin synthesis and alkaline phosphatase activity. J Biomech 2000;33:45–51. [5] Klein-Nulend J, Veldhuijzen JP, van de Stadt RJ, van Kampen GP, Kuijer

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