Mechanobiology of mandibular distraction osteogenesis: experimental analyses with a rat model

Bone 34 (2004) 336 – 343 www.elsevier.com/locate/bone

Mechanobiology of mandibular distraction osteogenesis: experimental analyses with a rat model Elizabeth G. Loboa, a,b,* Tony D. Fang, c Stephen M. Warren, c Derek P. Lindsey, b Kenton D. Fong, c Michael T. Longaker, c and Dennis R. Carter a,b a

Biomechanical Engineering Division, Mechanical Engineering Department, Stanford University, Stanford, CA 94305, USA b Rehabilitation R&D Center, VA Palo Alto Health Care System, Palo Alto, CA 94304, USA c Department of Surgery, Stanford University School of Medicine, Stanford, CA 94305-5148, USA Received 12 February 2003; revised 8 September 2003; accepted 31 October 2003

Abstract We analyzed mechanobiological influences on successful distraction osteogenesis (DO). Mandibular distraction surgeries were performed on 15 adult male Sprague – Dawley rats. Animals underwent gradual distraction (GD), progressive lengthening by small increments (5-day latency followed by 0.25 mm distractions twice daily for 8 days followed by 28-day maturation period). Distracted hemimandibles were harvested on postoperative days (POD) 5, 7, 10, 13, and 41. Load-displacement curves were then recorded for ex vivo distractions of 0.25 mm and stresses determined. Histologically, new bone formation appeared in GD specimens on distraction day 2 (POD 7), filling 50 – 60% of the gap by distraction day 8 (POD 13), with nearly complete bony bridging at end maturation (POD 41). Average tensile strains imposed by each incremental distraction ranged from approximately 10% to 12.5% during distraction days 2 – 8 and were associated with bone apposition rates of about 260 Am/day. Because this GD protocol was previously determined to be optimal for DO, we conclude that strains within this range provide an excellent environment for de novo bone apposition. Distraction caused tissue damage in distraction day 2, 5, and 8 specimens as evidenced by distinct drops in the load/displacement curves. Taken together, our interpretation of these data is that daily distractions cause daily tissue damage which triggers new mesenchymal tissue formation. D 2003 Elsevier Inc. All rights reserved. Keywords: Mandibular distraction osteogenesis; Mechanobiology; Rat model; Mechanical testing; Mesenchymal tissue

Introduction Although limb lengthening by distraction osteogenesis (DO) was first described in 1905 [8], the technique did not gain wide acceptance until Gavril Ilizarov [16 – 18] identified the physiologic and mechanical factors governing successful regenerate bone formation. In 1973, using a canine model, Snyder et al. [35] adapted distraction principles to the membranous craniofacial skeleton. Snyder’s success ignited the field of craniofacial DO and created the momentum for numerous experimental surgical models [3,11,19,21]. These large animal models refined the tech-

* Corresponding author. Department of Biomedical Engineering, North Carolina State University, 433 Daniels Hall, Campus Box 7115, Raleigh, NC 27695-7115. Fax: +1-919-513-3814. E-mail address: [email protected] (E.G. Loboa). 8756-3282/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2003.10.012

nical principles of craniofacial distraction and led to the first human mandibular distraction therapy in 1992 [23]. Despite its success, mandibular DO was intermittently complicated by atrophic fibrous union in which the gap tissue is characterized by disorganized collagen bundles, islands of intercalary collagen, and rudimentary bone spicules. Histomorphological and ultrastructural analyses reveal that successful bone DO induces osteoid and bone formation without a substantial cartilaginous intermediate tissue. Furthermore, the tensile distraction across a surgical osteotomy creates nascent bone formation in a plane parallel to the applied tension vector. This new bone forms centripetally from the osteotomized bone edges toward the center of the distraction gap [34]. Clinically, distraction protocols are divided into the latency period (time period between osteotomy and initiation of distraction), the rate and rhythm of distraction

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(amount and frequency of movements), and the maturation period (period of time the patient is maintained in rigid external fixation). To improve outcomes, surgeons have attempted to empirically modify the latency, rate, rhythm, and maturation periods [1,4,9,10,12,22,36,39]. Molecular analyses of gene expression during successful (osseous tissue engineering and bony union) and unsuccessful (fibrous union) distractions led to the application of novel recombinant proteins and gene modified protocols [28,30,31]. New devices were designed to control distraction directions and deliver growth factors [14,15]. Understanding the mechanobiology of this procedure at the tissue level may reduce complications (e.g., fibrous union) and enhance bone regeneration through the novel application of mechanical stimulation to guide differentiation of multipotent tissue within the distraction gap. Previous investigations have attempted to analyze the effects of interfragmentary strain magnitudes and strain rates on distraction regenerates using both long bone and membranous bone distraction models [26,27,32,33,37]. Those investigations, however, usually only analyzed the regenerating tissue at one time point—the end of distraction. Alternatively, if more time points were analyzed, strain calculations did not take into account the new bone formation that occurred during the distraction period. As such, the mechanobiological processes regulating how bone regenerates before, during, and after distraction are still not fully understood. Some insight into the stress and strain fields applied to the regenerating tissue during experimental long bone lengthening procedures has been gained using finite element analyses. Utilizing an experimental model of a mouse tibial lengthening, we have correlated regions of hydrostatic stress and tensile strain within the tissue regenerate to formation of specific skeletal tissues based on a previously published mechanobiological tissue differentiation concept [5 – 7,13]. That concept proposes that cyclic hydrostatic pressure causes cartilage formation, high tensile strain leads to fibrous tissue formation, and a combination of hydrostatic pressure and tensile strain triggers fibrocartilage formation. Given an adequate vascular supply, low hydrostatic stress and tensile strain allow for the direct formation of bone, with mild hydrostatic tension accelerating the rate of bone formation. The finite element analyses performed for the mouse tibial lengthening study gave results consistent with the mechanobiological tissue differentiation concept [5]; however, the study was deficient in that the mechanical properties necessary to define the models were unknown and had to be assumed. These properties can only be determined with mechanical testing, hence mechanical testing of the multipotent mesenchymal tissue regenerate within the distraction gap was one focus of our current investigation. Additionally, we sought to better characterize the tissue strain and stress histories that are associated with successful DO and relate those histories to histological information on bone regeneration.

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Materials and methods Animals All experiments were performed in accordance with Stanford University Animal Care and Use Committee guidelines. Adult male Sprague Dawley rats weighing between 300 and 400 g were purchased from Simonsen Laboratories INC (Gilroy, CA). Animals were housed in a light and temperature-controlled environment and given food and water ad libitum. Surgery Surgical procedures have been previously described [25, 34,38]. Briefly, animals (n = 15) were anesthetized (20 mg/ kg Ketaset, 4 mg/kg xylazine, 0.5 mg/kg acepromazine maleate), given a preoperative dose of antibiotics (10 mg/ kg cefazolin), prepped with betadine, and the incisors were clipped. An incision was made over the right hemimandible, the masseter muscle was divided, and the mandible exposed. An osteotomy was performed between the 2nd and 3rd molar using a diamond disc and the kerf was carefully measured using digital calipers with an accuracy of 0.01 mm. Two Flexi-post pins were placed 4 mm anterior and posterior to the osteotomy. The muscle and skin were then closed in layers and a custom-made distraction device was fixed to the pins. The distraction device consisted of a paralleled Lewa Jackscrew and guide pin embedded in a methylmethacrylate mold. After fixation of the distraction device, the distance between the pins was measured (Fig. 1A). Distraction protocol The gradual distraction (GD) protocol was the same as a previously published protocol for successful bone formation following mandibular distraction in a rat model (Fig. 1B) [29]. This protocol was established because it gave the optimal results for successful osteogenesis and bony union when compared to many other experimental protocols with lesser or greater distraction increments per day. The protocol consists of a 5-day latency period after the initial osteotomy and distractor device fixation followed by 8 days of distraction and 28 days of maturation/consolidation when no distraction is applied. During the 8-day distraction period, the mandibles were distracted a total of 0.5 mm/day at the rate of 0.25 mm every 12 h. Three GD specimens (n = 3) were harvested at each of five time points: end latency [postoperative day (POD) 5], distraction day 2 (POD 7), distraction day 5 (POD 10), distraction day 8 (POD 13), and end maturation (POD 41) to attain a total of 15 (n = 15) GD specimens. Distraction measurements were taken at each distraction using calipers with an accuracy of 0.01 mm. The distance measured was the gap between the centers of the heads of

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residual tensile force within the specimen was measured and the stress relaxation was measured until it reached equilibrium. Once equilibrium was attained, the specimen was pulled apart 0.25 mm in 0.5 s and maintained at that displacement until reaching equilibrium again. The displacement was returned to zero and the load was allowed to reach equilibrium. The tensile displacement test was then repeated with the same protocol. If there was at least a 10% decrease in the maximum force created in the second displacement test, then we assumed that tissue damage must have occurred during the first displacement test. Tissue processing and histology

Fig. 1. (A) Hemimandible osteotomy and location of distractor device (adapted from Bouletreau et al. [2]). (B) Mandibular DO protocol for the rat model. (C) Schematic demonstration of the mechanical testing apparatus.

two pins fixing the external distraction device to the right hemimandible (Fig. 1A). Measurements were taken three times before each distraction. The average pre-distraction distance was calculated as the average of the three measured values. The mandible was then distracted and post-distraction measurements were taken three times. The average post-distraction distance was calculated as the average of the three measured values. Actual distraction distance was defined as the average post-distraction distance minus the average pre-distraction distance. Mechanical testing Upon sacrifice, the right hemimandible was harvested with the distractor in place, scanned via computed tomography, and then kept frozen at 20jC until thawed at room temperature in a saline bath before mechanical testing. For mechanical testing, the anterior and posterior portions of the right hemimandible were potted in polymethylmethacrylate while the regenerate within the distraction gap was kept moist by gauze drenched with saline solution. Once potting was complete, each specimen was mounted in a customdesigned tensile testing machine. The machine incorporates a 1-kg load cell and linear displacement actuator horizontally mounted to measure forces from an imposed 0.25-mm tensile displacement of the specimen (Fig. 1C). The displacement was controlled by a motion controller connected to a laptop computer. Upon removal of the distractor,

After mechanical testing of the GD specimens, the mandibles were fixed in 4% paraformaldehyde, demineralized with Formical, processed via standard protocol, and embedded in paraffin. Specimens were sectioned axially, inferior to superior, every 5 Am and hematoxylin and eosin staining was performed using standard protocols [24]. Photomicrographs were taken with a digital camera. Histomorphometry was obtained using SPOT software. An experienced pathologist reviewed all slides in a blinded and independent fashion. Fifty slides for each specimen were analyzed for estimating the average width of new bone formation and average new bone percentage within the distraction gap. Briefly, for the average width of new bone formation, each side of the osteotomy front was used as the reference line. Linear distances between osteotomy fronts and new trabecular bone front from superior, center, and inferior portions of the specimens were measured on both sides of the osteotomy front. Average distance on each side was calculated and the combined average of the two was defined as the average width of new bone formation for the slide. All 50 slides for the specimen were analyzed in the same manner, and their average defined as the width of new bone formation for the specimen. For average percentage of new bone formation, the total area of new bone on each side of the osteotomy front was measured, as well as the total gap area between the two osteotomy fronts. The ratio between the total area of new bone and total gap area was calculated. The average of the 50 slides was defined as the average new bone percentage for the specimen. Calculation of tensile stresses and strains Tensile stresses were calculated using the force readings obtained from mechanical testing and cross-sectional areas (CSAs) measured assuming a rectangular cross-sectional geometry of the soft tissue regenerate. For CSA calculations, the superior– inferior and medial – lateral dimensions of the central distraction gap tissue were each measured 3 times with digital calipers that had an accuracy of 0.01 mm. The average of each dimension was calculated and these average medial – lateral and superior –inferior measurements were multiplied by each other to obtain the CSA of each

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specimen. Maximum tensile stress was calculated using the maximum distraction force reading resulting from the first 0.25 mm tensile displacement test (rmax = Fmax/CSA) and equilibrium tensile stress was calculated using the equilibrium force reading from the first test (req = Feq/CSA). ‘‘Nominal’’ average tensile strains in the gap were calculated using displacement measurements taken during each distraction (see Distraction protocol for GD specimens above) and incorporating the increases in gap size resulting from the distraction procedure. Average tensile strains in the soft regenerate gap tissue were calculated using displacement measurements taken during each distraction while incorporating both: (1) the increases in gap size resulting from the distraction procedure; and (2) the decreases in gap size associated with de novo bone formation within the gap. Therefore, at each time point, we calculated the gap size = displacement measurement (initial osteotomy gap + sum of distractions) average width of new bone formation. New bone formation within the gap was determined from histological and histomorphometric analyses of the specimens at each time point (see Tissue processing and histology above).

Results The operative procedures were well tolerated by all animals. There were no intra-operative or postoperative deaths. Twenty-seven rats were used in this study and 15 were analyzed. Twelve animals were eliminated from the study: 2 were removed due to soft-tissue infections and 10 were excluded because the distraction devices were dislodged during handling. Histology, histomorphometry POD 5 Specimens exhibited an intense inflammatory cell infiltrate within the osteotomy gap and surrounding soft tissues at this time point (Fig. 2A). Chondrocytes and new bony

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trabeculae were located along the periosteal edges of the osteotomies. The distraction gap contained disorganized collagen, but no new bone formation. POD 7 Specimens had now been distracted for 2 days. The inflammatory infiltrate was resolving and what appeared to be mesenchymal cells were migrating into the distraction gap (Fig. 2B). There was persistent periosteal bone formation and cartilage remained present along the periosteal edges of the osteotomies of GD specimens. We observed 8.3 F 0.09% of the distraction gap to be filled with new bone formation extending from the osteotomy edges towards the center of the distraction gap. Collagen fibers became thicker and more organized. POD 10 – 13 On distraction day 5 (POD 10), the inflammation had resolved in GD specimens. A marked increase in what appeared to be mesenchymal cells and extracellular matrix was noted at the center of the distraction gap (Fig. 2C). Collagen fibers were beginning to align along the axis of the distraction vector. New bone formation, without significant chondrocyte precursors, filled 31.6 F 9.8% of the distraction gap area. By the last day of distraction (POD 13), 54.9 F 11.7% of the distraction gap contained new bony trabeculae (Fig. 2D). Trabecular osteoblasts were enlarged and appeared active with abundant euchromatin. Large numbers of mesenchymal cells were still present at the center of the distraction gaps. All collagen fibers were aligned along the vector of distraction. POD 41 New bone was bridging the distraction gaps by this time point, the end of maturation (Fig. 2E). Some areas along the osteotomy edge and at the periphery of the hard callus were losing their trabeculation and becoming more cortical in appearance. Neovascular channels entirely bridged the osteotomy interface and, in numerous areas of the new

Fig. 2. Hematoxylin and eosin staining of the GD specimens (20 ). A = End of latency (POD 5); B = Distraction day 2 (POD 7); C = Distraction day 5 (POD 10); D = Distraction day 8 (POD 13); E = End of maturation (POD 41). AT = Anterior; PT = Posterior; LT = Lateral; MD = Medial. Arrows = osteotomies; arrowheads = new trabecular bone; (*) = mesenchymal tissue.

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cortical bone, the channels were coalescing to form mature Haversian systems. There was no evidence of chondrocytes or cartilage at this final time point and new bone filled 88.1 F 10.9% of the distraction gap. Tissue testing, distraction measurements, and tensile stress/ strain calculations The total amount of distraction achieved at the end of the 8-day distraction protocol was 3.36 mm with daily distraction averages equal to 0.42 mm/day. Nominal distraction strain (which does not incorporate the new bone formation within the gap) varied from 27% at the first distraction to 5.5% at the final distraction (Fig. 3A). Histological analyses of the specimens at each of the five time points analyzed showed that de novo bone formation began to occur after the end of latency (POD 5) and continued throughout distraction and maturation (Figs. 2A –E). We plotted average bone apposition in the distraction gap (Fig. 3B), and it showed that the end latency specimens (POD 5) exhibited almost no new bone formation, while specimens from distraction days 2, 5, and 8 (PODs 7, 10, and 13) all exhibited new bone within the distraction gap. For this plot, we assumed a linear change in new bone formation between the time points when de novo bone formation was directly calculated from histologic analyses (Fig. 3B). By the end of maturation (POD 41), the distraction gap was almost completely filled with new bone. We found that the highest bone apposition rate occurred between distraction day 2 (POD 7) and distraction day 8 (POD 13), followed by the time period

between end latency (POD 5) and distraction day 2 (POD 7) (Fig. 3C). By combining information about de novo bone formation at each time point (Figs. 2A – E and 3B) along with distraction measurements, we calculated the average distraction strain of the soft regenerate tissue in the gap (Fig. 3D). Similar to the nominal average distraction strain, this strain calculation included the increased distraction gap width resulting from the distraction protocol. However, it also considered the decrease in gap width resulting from de novo bone formation (Figs. 2A –E and 3B). The average distraction strain varied considerably from the nominal average distraction strain. While the nominal strain appeared to continually decrease throughout distraction (Fig. 3A), the true strain dropped to a strain of 12.5% by distraction day 2 (POD 7) and stayed within the range of approximately 10– 12.5% throughout the remainder of the distraction period (Fig. 3D). Mechanical testing Tensile forces associated with each distraction exhibited a viscoelastic relaxation response after the initial maximum distraction force was achieved (Fig. 4A). Maximum distraction forces ranged from a low of 0.01 N for an end latency specimen to a high of 6.65 N for a distraction day 8 specimen (Fig. 4B). After 5 days of distraction (POD 10), there was a substantial increase in the force required to perform the distraction (Fig. 4B). Distraction stresses within the distraction gap tissue were calculated assuming a uniaxial load from the distraction

Fig. 3. (A) Nominal average strain resulting from each distraction. Strain calculation only includes changes in the gap width associated with each distraction. (B) Average bone apposition within distraction gap. Data points are from histological analyses of new bone formation from both sides of the osteotomy fronts in end latency, distraction days 2, 5, and 8, and end maturation specimens. Between these time points, we assume a linear change in new bone formation (line). (C) Average bone apposition rates throughout distraction period. The new bone formation was from both sides of the osteotomy fronts. (D) Average strains of soft tissue regenerate as a result of each distraction. This strain calculation includes increases to gap width associated with distraction and decreases to gap width associated with de novo bone formation.

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Fig. 4. (A) Tensile force resulting from 0.25 mm distraction for typical distraction day 8 (POD 13) specimens. (B) Tensile forces resulting from a 0.25-mm displacement applied to all specimens. (C) Average cross-sectional areas (CSAs) of the tissue regenerate within the distraction gap throughout the GD protocol. Line is average CSA for each time point with a linear interpolation between points. (D) Tensile stresses resulting from a 0.25-mm displacement applied to all specimens.

forces (Fig. 4B) that was divided by the average minimum CSA of the tissue regenerate (Fig. 4C). The average CSAs increased noticeably with the initiation of distraction at the completion of the 5-day latency period. They appeared to reach a peak with an average area of 49.2 mm2 for the distraction day 5 (POD 10) specimens. By the end of distraction, that is, distraction day 8 (POD 13), the average CSA had decreased to 42.6 mm2 and remained close to this value through the end of maturation (POD 41). Mean distraction stresses (Fig. 4D) exhibited, as expected, a similar trend to distraction forces (Fig. 4B) with distraction stresses remaining fairly constant until distraction day 5 at which point an increase in distraction stress from distraction Table 1 Tensile displacement test results Time points End of latency

Specimens 1st 90% 1st 2nd Tissue Distraction Distraction Distraction damage force (N) force (N) force (N) (Yes/No)

#1 #2 #3 Distraction #4 day #2 #5 #6 Distraction #7 day #5 #8 #9 Distraction #10 day #8 #11 #12

0.01 0.88 0.12 0.01 0.97 0.03 0.17 0.94 1.25 4.44 0.58 6.65

0.009 0.792 0.108 0.009 0.873 0.027 0.153 0.846 1.125 3.996 0.522 5.985

0.01 0.46 0.11 0.01 0.79 0.02 0.16 0.25 0.94 2.09 0.23 1.89

No Yes No No Yes Yes No Yes Yes Yes Yes Yes

If 2nd distraction force is less than 90% 1st distraction force, then tissue damage is assumed to have occurred. The variations in testing results were most likely due to variations of individual experimental specimens, in general, and did not closely correlated with histology.

day 5 (POD 10) to distraction day 8 (POD 13) was noted (Fig. 4D). The tensile displacement tests were repeated to determine if tissue damage occurred as a result of each distraction. Of the three end latency specimens, only one exhibited tissue damage as determined by a maximum distraction force measurement for the second displacement that was less than 90% of the maximum distraction force for the first displacement (Table 1). Distraction day 2 (POD 7) and distraction day 5 (POD 10) specimens had two out of three specimens exhibit damage. All distraction day 8 (POD 13) specimens exhibited tissue damages. Therefore, it appeared that, in general, initiation of distraction did not cause tissue damage of the compliant tissue regenerate present within the distraction gap at the end of the 5-day latency period (POD 5). However, as time progressed, it appears that damage was imposed during each distraction.

Discussion The GD specimens exhibited new bone formation within the gap throughout distraction (PODs 7, 10, and 13) and achieved complete bony bridging by the end of maturation (POD 41). The distraction protocol used for the specimens, and particularly the magnitude of the incremental distractions, was selected as being particularly successful in bone regeneration compared to a wide range of other candidate protocols [33]. In the study reported here, we found that the highest rate of bone regeneration occurred between distraction day 2 (POD 7) and distraction day 8 (POD 13). While distraction forces and stresses appeared to vary considerably during this time period, average tensile strains dropped to

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12.5% per distraction by the end of distraction day 2 (POD 7) and remained between approximately 10 – 12.5% per distraction from distraction days 2– 8 (PODs 7– 13). We propose that tensile strains within this range triggered a high rate of new bone regeneration, and therefore may be particularly beneficial for bone regeneration. When distraction was concluded on POD 13, bone regeneration continued through the end of maturation at a much slower rate. These data are consistent with findings from other studies we have performed. In a mouse osteotomy healing model, we found that it takes 3 weeks for a 1-mm osteotomy to heal (unpublished data). In our current study, complete bony bridging was achieved in 6 weeks for the 4-mm displacement. On the basis of our experience, when comparing fixed osteotomy (i.e., ‘‘surgical fracture’’) healing to our DO data in this study, we feel that DO accelerates bone apposition. In our average distraction strain calculations, we incorporated changes in distraction gap width as a function of both distraction and new bone regeneration to arrive at strain values that incorporated both of these parameters (Fig. 3D). By including gap changes resulting from both distraction and de novo bone regeneration in our more precise strain calculations, we arrived at a fairly consistent range of strains during the period of peak bone regeneration from distraction day 2 (POD 7) through distraction day 8 (POD 13) that was not seen when calculating strains as a function of distraction alone. The magnitudes of average tensile strains reported here (approximately 10 –12.5%) would cause tensile failure in mature bone and cartilage. By performing our tensile tests twice, we found that tissue damage occurred in distraction day 2, 5, and 8 (POD 7, 10, and 13) specimens. Although the histological findings did not show specific morphological hallmarks associated with this damage, our mechanical testing data still suggest that the distraction protocol did cause tissue damage in vivo. This damage may have initiated continued recruitment and/or proliferation of mesenchymal tissue within the distraction gap during the distraction period. Histologic analyses of gradually distracted mandibles support this interpretation as mesenchymal tissue was observed in these mandibles during active distraction (POD 5 – 13). Our conclusion that tissue damage occurred was suggested by the results of our mechanical testing. In the future, further validation of this damage could involve the identification of a definitive biological mechanism, for example, the stimulation of an inflammatory response or release of tissue products related to damage, such as reactive oxides. Our interpretation of our findings that tissue damage occurs as a result of the daily step distraction protocol is supported by other investigators. Kessler et al. [20] studied the effects of continuous vs. noncontinuous distraction during mandibular DO in a pig model. By comparing bone regeneration from the application of an immediate, noncontinuous 1.5 mm daily distraction to that from a continuous 1.5 mm daily distraction (daily distraction applied gradually

over the course of 24 h), they concluded that noncontinuous distraction ‘‘results in a microtrauma in the soft tissues in the distraction zone. Vessels are disrupted and micro-hematomas are formed. The healing process is interrupted and must restart after each activation of the distractor.’’ The results of this study provide us with important information regarding successful bony differentiation during mandibular DO. We have determined the tensile forces, displacements, stresses, and strains occurring throughout distraction and defined strain levels corresponding to high rates of bone regeneration. These findings provide a framework for better understanding the mechanobiology of successful DO.

Acknowledgments We thank Dr. E. Ranheim for his expertise on all histological interpretations. This study was supported by National Institute of Health RO1 grant DE13028 to Michael T. Longaker.

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