Expression of smooth muscle actin in cells involved in distraction osteogenesis in a rat model

Journal of Orthopaedic Research

ELSEVIER

Journal of Orthopaedic Research 21 (2003) 20-27 www.elsevier.com/locate/orthres

Expression of smooth muscle actin in cells involved in distraction osteogenesis in a rat model B. Kinner a

D.M. Pacicca b, L.C. Gerstenfeld b, C.A. Lee T.A. Einhorn b, M. Spector a,*

b92,

Departnient of Orthopaedic Surgery, Brigham and Women’s Hospital, Haruurd Medical School, M R B 106, 75 Franci,y Street, Boston, M A 02115, U S A Department of Orthopaedic Surgery, Boston Unicersity School of Medicine, Boston, M A 02118, USA

’

Received 28 September 2001; accepted 18 April 2002

Abstract

Distraction osteogenesis has proven to be of great value for the treatment of a variety of musculoskeletal problems. Little is still known, however, about the phenotypic changes in the cells participating in the bone formation process, induced by the procedure. Recent findings of the expression of a contractile muscle actin isoform, a-smooth muscle actin (SMA), in musculoskeletal connective tissue cells prompted this immunohistochemical study of the expression of SMA in cells participating in distraction osteogenesis in a rat model. The tissues within and adjacent to the distraction site could be distinguished histologically on the basis of cell morphology, density, and extracellular matrix make-up. The percentage of SMA-containing cells within each tissue zone was graded from 0 to 4. The majority of the cells in each of the zones stained positive for SMA within five days of the distraction period. The SMA-containing cells included those with elongated morphology in the center of the distraction site and the active osteoblasts on the surfaces of the newly forming bone. These finding warrant further investigation of the role of this contractile actin isoform in distraction osteogenesis and investigation of the effects of modulation of this actin isoform on the procedure. 0 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.

Introduction

Since its introduction into the clinic 30 years ago distraction osteogenesis has proven to be of substantial value in the treatment of a wide array of musculoskeletal problems [1,8]. However, our understanding of the cellular processes induced by the mechanical environment of distraction is incomplete. Insights into the phenotypic changes of the cells participating in the bone formation process could lead to improvements in the procedure as well as to a deepened understanding of the behavior of osteogenic cells. The fact that cytoskeletal proteins might be expected to play a role in the response of

*Corresponding author. Tel.: +I-617-732-6702; fax: +I-617-7326705. E-mail address. [email protected] (M. Spector). Permanent address: Department of Trauma Surgery, Clinics of the University of Regensburg, Germany. Current address: Department of Orthopaedic Surgery, Wake Forest University School of Medicine, Winston-Salem, NC 27 157.

’

’

musculoskeletal connective tissue cells to the distraction strains, and that a contractile actin isoform may be of importance in the osteogenic process has drawn attention to the cytoskeletal aspects of the phenotypic changes induced by distraction. Recent studies have identified a contractile muscle actin isoform, a-smooth muscle actin (SMA), in osteoblasts [12,17] as well as in a number of other musculoskeletal connective tissue cells [30]. Associated studies demonstrated the ability of SMA-expressing osteoblastic cells to contract a collagen-glycosaminoglycan analog of extracellular matrix in vitro [12,17]. It was proposed that SMA-enabled contraction might be responsible for the retraction of osteoblasts on the bone surface at the initiation of the remodeling cycle. Moreover, SMA-enabled contraction may allow for the generation of the higher forces required for the cellular modeling of the newly synthesized extracellular matrix, to impart the tissuespecific architecture. Thus, the elevation of SMA expression might be expected to be associated with an osteogenic process such as bone transport.

0736-0266/03/$ - see front matter 0 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved PII: SO7 3 6 - 0 2 6 6 (02)OOOS 8 - 8

B. Kinner et al. / Journal of Orthopaedic Research 21 (2003) 20-27

The effects of mechanical forces on the total actin content and expression of other cytoskeletal proteins in osteoblasts [ 161 and cells participating in distraction osteogenesis [ 181 have been investigated in recent studies in vitro and in vivo. This work was prompted in part by the recognized importance of the structural integrity of microfilaments for the signal transduction of mechanical stimuli within osteoblasts [16]. These observations raise the question of how mechanical strain specifically alters the expression of SMA in the muscuoskeletal connective tissue cells affected by distraction osteogenesis. Collectively these studies stimulated this investigation of the percentage and distribution of SMA-expressing cells induced by distraction osteogenesis in a rat model. A related objective was to determine the effects of ambulatory loading of the treated limb on the expression of SMA induced by the distraction procedure.

Material and methods Animal model and treatment groups

A total of 19 adult rats were used in this study that was approved by the institutional animal care committee at Boston University where the surgery was performed. The male Sprague-Dawley rats were 4 0 6 500 g and all under six months of age. The model that was used was the same as that recently described in the literature [23].Details of the procedure can be found in that paper [23]. In order to reduce the loading of the distracted limbs in selected groups of animals, a through-knee amputation was performed immediately following the index operation using a procedure that was previously described [23]. The animals were caged separately and allowed to roam their cages freely. All rats were given buprenorphine 0.05 mg/kg BID for three days post-operatively. After a latency period of two days, on postoperative day three, distraction was begun at the rate of 0.25 mm twice a day for up to 13 days (Table 1). Animals were allocated to one of four treatent groups (Table 1 shows the animals available for this study). Every group consisted of weight-bearing and non-weightbearing animals (Table 1). Animals in Group I received the osteotomy only and were sacrificed after two days as latency period controls (Table I).

r-Smooth muscle actin ininiunoRistochet~ii,~~ry

After sacrifice the whole femur was harvested and fixed in formalin. Specimens were decalcified and embedded in paraffin using conventional technique. Sections, 5-7 pm in thickness, were cut on a microtome and deparaffinized in preparation for hematoxylin and eosin staining and SMA immunohistochemistry. For SMA immunohistochemistry, a monoclonal anti-SMA antibody (product no. A-2547, Clone 1A4, Monoclonal Anti-a Smooth Muscle Actin, Sigma Chemical Co., St. Louis, MO) was used. This antibody binds to the amino terminal decapeptide of SMA [29]. We have been using this Sigma antibody in our studies for several years [20,21,26]. Moreover, it has been employed in many laboratories around the world for over 10 years in numerous investigations for immunolocalization of SMA in immunohistochemistry [4,5,13,24] and for Western blot analyses [5,13,25]. Deparaffinized hydrated sections were digested with 0.1% trypsin for 1 h at room temperature and treated with 3% hydrogen peroxide to quench endogenous peroxidase. The sections were subsequently treated with the monoclonal SMA antibody. After incubation with the primary antibody the slides were incubated with a biotinylated affinitypurified secondary antibody. Streptavidin peroxidase was used before processing the slides using the aminoethyl carbazole (AEC) chromogen kit (Sigma Chemical Co.). Mayer’s hematoxylin was used as counterstain. For each staining run, negative controls were included. The negative control section was stained with Myeloma IgG2,, diluted to the same concentration as that in the monoclonal antibody solution, instead of primary antibody. Cells were identified as SMA-positive based on the following criteria: the negative control displayed no distinct chromogen, the intensity of the chromogen in the cells of interest was comparable to that in the smooth muscle cells of the vessel walls, and the chromogen was restricted to the cytoplasm of the cells. Method of evaluation and statistical analyses

The central longitudinal microtomed section was selected for analysis. Proceeding from the middle of the distraction site in proximal and distal directions, the following zones were identified based on the following histological criteria, and each zone was evaluated separately: I Fibrous tissue in the center of the distraction site (Fig. l a x ) . This zone comprised fibrocollagenous tissue that often displayed a crimp (Fig. 1b) to the collagen matrix and fibroblasts of elongated and ovoid morphology. I1 A hypercellular region of neo-osteogenesis displaying newly forming osteoid processes (Fig. l a and d). This zone was distinguishable from the region comprising trabecular bone (Zone 111) by: the

Table 1 a-SMA expression durinr distraction osteogenesis. Grade for the percentage of cells containing 3-SMA--mean Group ( n )

L”

Db

S‘

Zones IV urox.

I WB(3) IA(2)

2 2

0

0

2 2

2.5 (2-3) 2

I1 WB (2) I1 A (2)

2 2

5 5

7 7

I11 WB (3) I11 A (3)

2 2

9.5 9.5

IV WB (2) IVA(2)

2 2

(range)

IIIU

Ilu

I

I1 distal

IIId

3 (0-3)

-

1

-

-

-

-

-

-

-

2 2

2.75 (2.5-3) 2

2 2.75 (2-3.5)

2.25 (2-2.5) 2.25(1-3.5)

2.25 (2-2.5) 2.5 (1.5-3)

2 2.25 (1-3.5)

2 2.5 (2-3)

2 2.5 (2-3)

12 12

2.5 (1.5-3) 3 (2.5-3)

3.25 (3-3.5) 3 (2Z3.5)

3.5 ( 3 4 ) 3.5 (2.5-3.5)

3 3 (3-3.5)

3 3.5 (2.5-3.5)

3 3

3 3

18 18

2.75 (2.5-3) 3

3 3

3.75 (3.54) 3.75 (3.54)

3 2.5

3.25 3.5 ( 3 4 )

3 (3-3.5) 2.75 (2.5-3)

2.75 (2.5-3) 2.5 (2-3)

~~

13 13

21

WB-weight-bearing; A-amputation. Latency period (days). ’Distraction period (days). Sacrifice time (days).

1Vd

~

22

B. Kinner et al. I Journal qf Orthopuedic Research 21 (2003) 20-27

Fig. 1. Micrographs of immunohistochemical SMA preparations of longitudinal sections through distraction sites. (a) Low magnification image providing an overall view of a typical distraction site showing the locations of Zones I, IT, 111, and IV. Group IV sample; animal # 5 ; weight-bearing ( C 4 o r t e x ) . (b) Micrograph of Zone I in an animal (#7) from Group IV that underwent amputation. The organized fibrous tissue often displayed a crimp (arrows) of the collagen fibers (scale bar: 40 pm). Inset, negative immunohistochemical control section that was not stained with the SMA antibody (scale bar: 40 pm). (c) Cells in Zone I of a sample from a Group I1 animal (#7) that underwent amputation. Vitually all of the elongated cells (red arrow) contained SMA. The cells with a rounded nucleus and plump cytoplasm that may have been leukocytes (black arrow) did not label with the SMA antibody. Large accumulations of red blood cells could be found in this zone in some of the specimens (white arrow). The red cells did not contain SMA (scale bar: 40 pm). (d) Sample of Zone I1 in a Group (IV) animal (#6) with amputation. Zone 1 tissue can be see at the left merging with Zone TI (at the right). Note the strings of cells oriented in the same direction running from Zone 1 into Zone 11, with a dramatic change in morphology from an elongated shape in Zone I (white arrow) to a more rounded appearance in Zone I1 (black arrow). Toward the right of the micrograph the rows of the rounded cells are separated by the newly formed osteoid (0)(scale bar: 20 pm). (e) Micrograph of the Zone I11 tissue in a Group (IV) amputated animal (#6). Finger-like processes of osteoid (0)took on a more defined shape, beginning to appear as trabeculae in this zone, and were wider than those that appeared in Zone 11. Some of the osteocytes dispersed throughout the matrix contained SMA (white arrow) while other did not (black arrow) (scale bar: 40 pm). (0Sample of Zone IV in a weight-bearing animal (#6) in Group 111. An area of mature trabecular bone along the periosteum is shown. At the top of the image are blood vessels (white arrow) defined by SMA-positive smooth muscle cells. Virtually all of the osteoblasts stain positive for SMA (black arrow). Some of the osteocytes in the periosteal bone and most of the osteocytes in the underlying cortical bone (C) do not contain SMA (scale bar, 100 pm).

B. Kinner et al. J Journal

of' Orthopaedic Research 21 (2003) 20-27

23

indication of the relative percentage of SMA-containing cells in each zone. An estimate of the cell number density in Zone I and I1 of all specimens was made by counting the cells with the use of a grid in the eyepiece of the microscope. Ten randomly selected areas in each zone were analyzed for this purpose. Selected groups were compared using a contingency table and Fisher's exact test and Kruskal-Wallis test for analysis of variance (ANOVA). Statistical analyses were performed using GraphPad [email protected] Software and Statview. Dunn's post-hoc test was used for comparison of individual time points. The criterion for statistical significance was p < 0.05.

Results

Fig. 1 (continued)

thinness of the osteoid processes; the absence of a defined trabecular shape to the osteoid processes; and a higher number density of plump cells in the interstices among the osteoid processes. 111 Trabecular bone with active osteoblasts (Fig. l a and e). demonstrating a preferred orientation along the longitudinal axis. IV Mature trabecular bone comprising thicker trabeculae than in Zone I11 (approximately twice the thickness) and a more uniform distribution of osteocytes (Fig. l a and f). This zone included the periosteal and endosteal new bone along the diaphyses. The width of the zones was measured using digitized images and Scion Image Analysis software. The percentage of cells staining for SMA within each zone was evaluated using the following grading scheme: 0 1 2 3

4

none trace, up to 10% 11-50'Xr 5 1- 9o'yn 91 10O'Yo

A ratio scale for the evaluation of the percentage of SMA-positive cells was not used because of the difficulty in accurately determining the number of cells due to the high cell number density and the overlap of cells in the histological sections. The above ordinal scale was based on pilot analyses of the data and was deemed to be useful for providing an

The longitudinal histological sections through the distraction site clearly revealed the osteotomized edges of the cortices (Fig. la). Also evident was the trabecular bone formed within the distraction site of the specimens at the 12- and 18-day sacrifice periods (Groups 111 and IV; Fig. la). The trabeculae appeared to emanate from the osteotomized surfaces. Of note was the preferred orientation of the trabeculae along the long axis of the femur (Fig. la). No such trabeculae were present in the latency period control specimens. A non-osseous zone at some location in the middle of the distraction site could be identified in all of the samples. Smooth muscle cells surrounding the vessels stained prominently with the chromogen (red), and this provided internal positive controls for the immunohistochemistry. The negative control sections, stained with the Myeloma IgG2, instead of the SMA monoclonal antibody, did not display noticeable labeling with the chromogen. Cells expressing SMA were prominent throughout all of the zones comprising the newly formed tissue in Groups 11,111, and IV, but not in Group I. There was an overall trend of increasing SMA expression with sacrifice time irrespective of the tissue make-up. Of importance was the finding that the percentage of cells containing SMA was noticeably higher in the distraction zones compared to the uninvolved osseous tissue a few millimeters away from the distraction site, in both proximal and distal directions. Each of the four histological zones (Fig. la) comprising the distraction region displayed characteristic features that allowed them to be distinguished in tissues from each of the treatment groups. The widths of Zone I for Groups 11, 11, and IV were 3 4I 0.7 (mean fSEM), 4 It 0.3, and 4.3 4I 0.6 mm, respectively. The width of Zone I1 was approximately 0.2 mm for all groups. This smaller width precluded a confident evaluation in each of the specimens. For Zone I11 the widths for Groups 11, 11, and IV were 1 .2 3 ~ 0 .62.1 , f 0 . 4 , and 3.5410.7 mm, respectively. Table 1 shows the grading for the percentage of SMA-containing cells within each of the zones.

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B. Kinner et rrl. I Journul of Ortkopaedic Research 21 (2003) 20-27

Fibrous tissue (Zone I )

A prominent cellular tissue, fibrous in nature, could be found at the center of the distraction site in all of the groups. This tissue was readily distinguishable from the newly forming bone in Zones II and 111. Two-factor ANOVA demonstrated a significant effect of distraction period on the smaller width of Zone I (p = 0.003), comparing Groups IT, 111, and IV, but no significant influence of weight-bearing on the width of this zone (p = 0.3). The tissue in this zone in the two day control specimens (Group I) was comprised of rounded cells in an avascular fibrin network. Elongated cells aligned along the longitudinal axis of the bone could also be seen dispersed through this zone in the two day control. There was prominent SMA staining of approximately 30% of the rounded and elongated cells. In the earliest distraction group (Group II), fibroblast-like cells were distributed loosely throughout the distraction gap. The elongated cells in Zone I were aligned along the long axis of distraction and appeared to stream from trabecular or endosteal surfaces to form the fibrous tissue comprising this zone. There was a predominance of elongated cells most of which contained SMA (Fig. 1c). Rounded cells were still present but most of these did not label with the SMA antibody (Fig. lc). Large accumulations of red blood cells could be found in this zone in some of the specimens (Fig. lc). Higher magnification of the elongated cells demonstrated that in many the SMA appeared to be staining prominent stress fibers. The density of the fibrous tissue increased with distraction time, when comparing Groups I1 and Ill. During the consolidation phase (Group IV), the fibrous tissue demonstrated a more organized appearance, displaying a crimped structure to the collagen fibers (Fig. 1b). The period of this crimp 22 1 pm ( n = 7; mean i SEM; Fig. lb), was similar to the measurement recently reported for human meniscus [2]. The large majority of cells in this region in the distracted groups (Groups II, 11, and IV) contained SMA (Table 1). Some heterogeneity of staining was found, with a tendency of more intensive staining in the center, compared to the periphery of distraction site. There was a trend of increasing SMA expression with distraction time. When comparing Groups I (control) and II (early distraction) with Groups 111 and IV, a significant difference was found with a greater percentage of SMAcontaining cells at the longer treatment periods (MannWhitney test, p = 0.009).

*

Newly forming osteoid projections (Zone 11)

There was a distinct transition zone (Zone II; Fig. Id) between the fibrous tissue of Zone I and the formed trabeculae of Zone I l l . Continuous strings of cells,

highly oriented longitudinally, ran from Zone I into Zone 11 (Fig. Id). A dramatic change in morphology of the cells from an elongated shape in Zone I to a more rounded appearance in Zone I1 (Fig. Id) was evident. Rows of the rounded cells began to be defined by separations, that deeper into Zone II became osteoid (Fig. Id). Rounded cells that were detached from the strings of cells (proceeding towards Zone 111) became surrounded by osteoid and were contained with lacunae (Fig. Id). The majority of the elongated and rounded cells in this zone contained SMA. The thin processes of matrix in Zone I1 were continuous with the immature bony trabeculae in Zone 111, and the Zone 111 trabeculae continuous with the mature trabeculae in Zone IV. This was consistent with the identification of the processes in Zone II as newly formed osteoid and the associated cells osteoblasts. The average cell density in Zone II was 6600 & 1100 cells/mm2 compared to 3800 i 1700 cells/mm2 in Zone I, a highly significant difference (Wilcoxon signed rank test, p = 0.004). As in Zone I, a high percentage of SMA-positive cells could be found in Zone 11, with a similar trend of increasing expression of SMA with time (early vs. late; Table 1; Mann-Whitney, p = 0.009). Newly jorming trubecular bone with active osteoblasts (Zone 111)

This zone generally extended from the edge of the osteotomy into the distraction gap to Zone 11. Particularly in the Group IV specimens, the finger-like processes of osteoid in Zone I11 were wider than in Zone I1 and the clearly arranged strings of rounded cells found in Zone 11 were not present in Zone I l l (Fig. le). Instead, osteocytes were dispersed throughout the matrix (Fig. le). The trabecular bone surface was lined predominantly by active osteoblasts (viz., plump cells), of which a high percentage stained positive for SMA (approximately 70%, corresponding to an average score of 3). Cells enveloped by their newly formed matrix also contained SMA. However, with increasing sacrifice time, SMA expression of these osteocytes decreased. As observed in the other zones there was a significantly greater expression of SMA in the later groups (Ill and IV) when compared to Groups I and II (Table 1; MannWhitney, p = 0.04). Mature trubecular bone (Zone I V )

This zone extended from the edge of the osteotomy into the intact bone, proximal and distal to the distraction gap (Fig. I f ) . These areas of mature trabecular bone principally comprised periosteal bone formation along the proximal and distal bone fragments. There were, however, occasional regions of trabecular bone

B. Kinner et al. I Journal of Ortlzopaedic Reseurch 21 (2003) 20-27

within the medullary canal and along the endosteal surface. These mature trabeculae were distinguished from the newly forming trabeculae in Zone 111 on the basis of: (a) the branching shape of the trabeculae, (b) the vascularization of the forming marrow spaces, (c) the uniform distribution of osteocytes, (d) osteoclastic activity, and (e) the uniform spacing of active (plump) and resting (flattened) osteoblasts on the bone surface (Fig. If). Of interest was that SMA was contained in some cells near the bone surface, which displayed prominent stress fibers. Cartilage formation was only seen occasionally during the lag-phase and early distraction phase. SMA could be found in approximately 30% of the chondrocytes, which displayed the typical spherical morphology and were located within lacunae. There was marked staining, however, of the osteoblasts lining the trabecular bone surfaces with the SMA antibody. Approximately 70% of the bone lining osteoblasts contained SMA (Fig. 10. Within our time frames and animals available there was no difference in SMA expression for the different time points (Table 1; Kruskal-Wallis p = 0.1) or comparing groups I and I1 with I11 and IV (Mann-Whitney p = 0.09). Aqacent uninvolved bone In a few specimens the metaphyseal bone of the adjacent femoral condyles or the proximal tibia1 was available for evaluation of the SMA expression. Overall SMA expression of the uninvolved bone was
Discussion

This is the first report of the expression of a contractile muscle actin, SMA, in cells in the tissues formed during distraction osteogenesis. There was a consistent finding of a significant percentage of fibroblasts containing SMA in the fibrous tissue in the center of the distraction gap, in the osteoblasts responsible for the formation of the newly forming projections of osteoid, and in the osteoblasts lining the trabecular bone surfaces in the consolidation phase. Approximately 30% of the chondrocytes in the cartilage that was occasionally seen also contained SMA. Of interest was the fact that this contractile muscle actin was found in cells with greatly differing morphologies and functions. These findings confirm prior demonstration of SMA expression in these cell types in other intact and injured musculoskeletal tissues. The finding of SMA in the elongated and sometimes crimped fibroblast-like cells of the zone in the middle of the distraction gap recalls the

25

SMA-expressing dermal fibroblasts (myofibroblasts) in skin wound healing and in Dupuytren’s disease [28] and SMA-containing ligament cells in recent work [14,21]. The observation of SMA in bone lining cells confirms a previous report examining canine and human bone samples [17]. Of importance are the related in vitro studies that have demonstrated that SMA-expressing ligament [ 151 and tendon [27] fibroblasts and osteoblasts [171 can contract a collagen-glycosaminoglycan analog of extracellular matrix. The findings of the current investigation are generally in accordance with the supposition that all musculoskeletal connective tissue cells have the capability to express this actin isoform by virtue of its well-documented expression in the putative progenitor cell, the mesenchymal stem cell (viz., the bone marrow stromal cell) [30]. It has been proposed that the capability to express SMA is a trait conferred to musculoskeletal connective tissue cells by their progenitor, the mesenchymal stem cell. Expression of SMA in these connective tissue cells is up- and down-regulated by certain regulators released during physiological and pathological processes [30]. While the widespread expression of SMA in connective tissue cells is now being reported, there remains uncertainty about its role. It has been proposed that SMA-enabled cell contraction generates the higher forces required of cells to model newly forming extracellular matrix and to impart architectural features such as crimp to the tissue [30]. This may be one of the roles that SMA expression plays in distraction osteogenesis. However, further work will be required to clearly demonstrate its role in this clinically important treatment modality. While the results clearly demonstrated the presence of SMA in a large percentage of the different cell types engaged in distraction osteogenesis, the limited sample size did not fully allow for the tracking of the time course of SMA expression. It could be demonstrated, however, that there was a significant prevalence of SMA expression in all cells participating in distraction osteogenesis when compared to the uninvolved bone and soft tissue in the same specimens. The suggestion there might be a significant difference in SMA expression in cells at the early and late stages of distraction osteogenesis needs further investigation. The positive finding of SMA in such a large percentage of cells warrants study of the stimulus of the expression of this contractile actin isoform. Is expression induced directly by the action of the distraction forces on the cells or due indirectly to the release of soluble regulators by the cells? Several studies have demonstrated the myriad growth factors expressed during distraction osteogenesis. One of those expressed in high levels [22], transforming growth factor (TGF)-Pl, is a known regulator of SMA expression. Using a cellseeded collagen gel model, it was shown that TGF-P but

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B. Kinner et al. I Journal of Orthopaedic Research 21 (2003) 20-27

not platelet derived growth factor, fibroblast growth factor, and epidermal growth factor, markedly enhanced the ability of several types of fibroblast to contract [19]. Later studies [6,25] indicated that TGF-PI might also be able to promote the differentiation of fibroblasts into myofibroblasts, demonstrating that TGF-P1 induces expression of SMA in fibroblastic cells in vivo and in vitro [6]. More recently, it was shown that TGF-PI enhanced the SMA expression in rat and mouse mesenteric wounds [9]. Collectively these studies provide rationale for the hypothesis that the distraction procedure up-regulates SMA expression through the action of TGF-fi1. The direct testing of this hypothesis needs to be considered in future work. The rat has been proven to be a useful animal model to study distraction osteogenesis [3]. Our findings of the chronology of healing in this rat model are similar to those previously reported [ 101. Fibroblast-like cells filled the gap with collagen bundles. Osteoid projections and woven bone appeared to be laid down on these collagen scaffolds, and newly formed vascular sinuses appeared to be the sites from which bone formation was initiated within the distraction gap. Yasui et al. [31] found that endochondral bone formation was prominent only in the early stage of distraction, but intramembranous bone formation became the predominant mechanism of ossification at later stages. Jazrawi et al. [lo] found that endochondral bone formation was only present peripherally but not in the distraction gap, unless the distraction was discontinued. This animal model proved to be valuable in tracking the association of SMA expression and the cellular composition of the tissues involved in the bone transport procedure. It will be useful in future work to further identify the matrix molecules in the various distraction tissues (i.e., in Zones 11, 111, and IV). The role of weight bearing during distraction osteogenesis is of great interest. However, the literature as well as our current data do not allow for a general suggestion how to proceed clinically. We could neither see an effect on the amount of newly formed bone [7,11] nor a clear effect on vessel formation as previous data might have suggested. There was also no effect on SMA expression. The present study demonstrates the widespread expression of SMA in cells participating in distraction osteogenesis. These findings lay the foundation for future work investigating the various roles of this contractile actin isoform in distraction osteogenesis. Such investigations may inform future therapeutic approaches to improve this important treatment modality. Acknowledgements The authors are grateful for the histological assistance of Sandra Zapatka Taylor.

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