Bone 41 (2007) 437 – 445 www.elsevier.com/locate/bone Fracture repair: Modulation of fracture-callus and mechanical properties by sequential application of IL-6 following PTH 1–34 or PTH 28–48 Nimrod Rozen a,b,1 , Dina Lewinson a,b,⁎, Tova Bick b , Zvi C. Jacob a,c , Haim Stein c , Michael Soudry b,c a Department of Anatomy and Cell Biology, The Rappaport Family Faculty of Medicine, Technion-Israel Institute of Technology, PO Box 9649, Haifa 31096, Israel b Institute for Research of Bone Repair, Department of Orthopaedic Surgery A, Rambam Medical Center, 8 Ha'aliya str., PO Box 9602, Haifa 31096, Israel c Division of Orthopaedics, Rambam Medical Center, 8 Ha'aliya str., PO Box 9602, Haifa 31096, Israel Received 24 January 2007; revised 19 April 2007; accepted 19 April 2007 Available online 8 May 2007 Abstract Fracture healing presents a sequence of three major stages: inflammation and granulation tissue formation, callus formation and remodeling. Our working hypothesis was that fracture-repair might be enhanced by stimulating proliferation of chondrocytes and osteoblasts in the early stages of fracture healing followed by sequential acceleration of the remodeling process. In the present study we employed a novel device developed by us implementing a standardized fracture in rat tibiae. We investigated the effect of PTH 28–48 or PTH 1–34 alone or in sequence combination with IL-6 together with its soluble receptor (IL-6sR) on fracture repair. PTH 28–48 or PTH 1–34 was applied locally into the hematoma of fractures on days 4, 5 and 6 and IL-6+ its soluble receptor on days 7, 9, and 11. Post-fracture callus volume as measured 14 days post-fracture was increased significantly only by PTH 1–34 (20%; P b 0.01). When one of the PTH fragments and IL-6 + IL-6sR were applied sequentially callus volume was increased significantly (33%; P b 0.01). X-rays radiography at 5 weeks post-fracture showed enlarged callus volume following treatment by either PTH fragments alone, and complete union following the sequential injection of both PTH fragments and IL-6 + IL-6sR, only. Only the combination of one of the PTH fragments with IL-6 + IL-6sR, as measured 6 weeks post-fracture by three point bending, changed dramatically the quality of the regenerating bone as presented by a 300% increase in mechanical resistance when PTH 1–34 was combined and 200% when PTH 28–48 was combined relative to vehicle-treated fractured bones. We conclude that the sequential application of IL-6 + IL-6sR with both PTH fragments has the potential of enhancing fracture healing in long bones and should be further explored in preclinical and in clinical studies. © 2007 Elsevier Inc. All rights reserved. Keywords: Fracture repair; Bone; IL-6; PTH 1–34; PTH 28–48 Introduction Fracture healing after injury in a long bone is a well regulated multi-step process; however, 5–10% of the fractures are associated with non-union or delayed healing [1]. The interest in the clinical applications of growth factors and other peptides as enhancers of bone repair has stimulated several studies ⁎ Corresponding author. Institute for Research of Bone Repair, Orthopaedic Surgery A, Rambam Medical Center, 8, Ha'aliya st., Haifa 31096, Israel. Fax: +972 48543606. E-mail address: dinal@tx.technion.ac.il (D. Lewinson). 1 Present address: Department of Orthopaedic Surgery, Ha'Emek Medical Center, Afula 18101, Israel. 8756-3282/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2007.04.193 resulting in as yet no definitive conclusion and pragmatic recommendations [2]. One of the agents studied intensively on its potential effect on fracture healing is the parathyroid hormone (PTH) and specifically its amino terminal fragment PTH 1–34. PTH has two opposite effects on bone if administered in different ways. While continuous administration causes bone loss, intermittent injection increases bone volume and bone density [3–5]. The primary target of PTH in bone is the osteoblast which expresses a single PTH receptor (PTH1R), which is a common receptor also to the hypercalcemia associated peptide PTHrelated protein (PTHrP) [6]. The complexity of PTH skeletal actions, catabolic as well as anabolic has made their evaluation for the most part difficult to interpret. However, these ambiguous actions of PTH on bone suggest its effect as a bone turnover 438 N. Rozen et al. / Bone 41 (2007) 437–445 Fig. 1. Fracture scheme demonstrating the four sites (marked as bold squares), that were chosen for the histomorphometric analysis. Sections that displayed a fracture gap between 4 cortices were chosen for analysis. Two of them were located under the periosteum proximal and distal to the fracture gap and the other two adjacent to the cortices at the fracture gap. Four Alcian blue-H&Estained sections from each callus, approximately 50–100 μm apart, were analyzed histomorphometrically. Surface areas occupied by fibrous tissue, cartilage, and cancellous bone were analyzed. Mean values ± SEM of areas of each type of tissue were calculated separately. modulator in fracture repair. Experiments in animal models have used intermittent application of various doses of PTH 1–34 in different orthopaedic applications. In a study in parathyroidectomized rats, PTH administration was shown to enhance early fracture-healing [7]. In a number of recent reports, doses ranging from 10 to 200 μg/kg in rat models of fracture-healing were found to be associated with substantial increases in both mechanical and histological properties [8–11]. Nakajima et al. [9] observed stimulation of osteoprogenitor cells as revealed by PCNA immunostaining in day 2 post-fracture in the periosteum of fractured rats treated daily with 10 μg/kg PTH. They also reported increased levels of mRNA expression of collagen typeI, osteonectin, alkaline phosphatase, and osteocalcin, all markers of differentiated osteoblasts during the early stages of the healing cascade and in a later stage (4–21 days post-fracture). In other studies, in which models of impaired bone metabolism were used, PTH analogs were shown to reverse the inhibition of bonehealing in ovariectomized rats and in rabbits treated with corticosteroids. In one report, PTH 1–34 was shown to increase bone in-growth and pullout strength in porous metallic implants [12–14]. Despite its promising potential in enhancing fracture repair, no results of trials have yet been published on the use of PTH 1–34 (Teriparatide) in clinical setting. Our laboratory studied the effect of the synthetic mid-region fragment of PTH, PTH 28–48, and confirmed in newborn mice previous in vitro observations, that it exerts anabolic effects on cartilage in vivo as well [15–20]. These effects are probably mediated through binding to the common PTH/PTHrP receptor and transduction of the signal by means of the protein kinase C pathway [21–23]. Moreover, It has been shown that PTH 28–48 up-regulates 1,25-dihydroxyvitamin D3 receptors in rat growth cartilages by the same pathway [24]. Interleukin-6 (IL-6) is a multifunctional cytokine that regulates pleiotropic biological activities in immune regulation, inflammation, hematopoiesis, and oncogenesis. Its effects are shared by other cytokines of the IL-6 family, such as leukemia inhibitory factor and oncostatin M [25,26]. It is also known as a stimulator of bone resorption [27]. IL-6 exerts its biological activities through interaction with specific receptors expressed on the surface of target cells. The receptor complex mediating the biological activities consists of two distinct membrane-bound glycoproteins: IL-6 receptor and gp 130, which is a signal transduction non-ligand binding component. Upon binding of IL-6 to the receptor, gp130 is homodimerized and is subsequently involved in down-stream signal processes binding tyrosine kinases and activating STAT1 and STAT3 transcriptional factors. Both IL-6R and gp130 also occur in soluble form in biological fluids and have been purified from human serum and urine [28]. Our group has previously demonstrated that systemic injection of IL-6 to nude (athymic mutant) mice enhances bone remodeling up to the point that the petrotic bone of the nude mice resembles that of a normal mice [29]. Therefore we decided to study the metabolic effect of IL-6 on bone remodeling during fracture repair and to explore whether its sequential combination with PTH fragments will benefit the fracture healing process without compromising the quality of the regenerated bone. Recently we have described the fracture healing cascade high-lightening important cross roads in which epigenetic intervention might enhance the healing process [1]. The present study presents an example applying this approach. Materials and methods Rat fracture model Wistar female rats (200–250 g) were used. Animals were maintained and sacrificed in a manner approved by the Institutional Committee for Animal Care Fig. 2. Histomorphometric analysis of 8 days (upper panel) and 14 days (lower panel) old fracture calluses. Sections from five rats were analyzed for each group. The histogram demonstrates surface areas ± SEM occupied by cartilage, woven bone and fibrous tissue. *P b 0.02 vs. control. N. Rozen et al. / Bone 41 (2007) 437–445 and Experiments of the Technion. Rats were anesthetized with intraperitoneal injections of sodium phenobarbital (4 mg/100 g body weight) (Rhöne Merieux Limited, Harlow, Essex, UK). A hollow wire pin (French 25) was introduced through the medial aspect of the tibia in the area of the ring of Ranvier until the middle of the medullary canal. Then, fracture was induced by the “Rozen device”, which was developed in our laboratory, to the mid-shaft of the tibia [30]. With this device the left tibia is clamped by two flexible screws proximally and distally to the mid-shaft and a closed fracture is induced by a direct external blow by means of a pendulum. The device administers controlled blows that produce anatomically uniform fractures. Following fracture the pin was pushed further until it reached the ankle joint. The right tibia remained untouched and served as an unfractured control for the same rat. Post operatively the fractured tibiae were immersed in an antiseptic solution in order to avoid infections. The rats were free to move in their cages with full weight bearing throughout the experimental period. All rats received the same Purina diet. Rats were divided randomly into experimental groups. Each experimental group contained 5–10 animals. Local application of PTH fragments and IL-6 On days 4, 5, and 6 post-fracture, at 11.00 AM, a volume of 0.2 ml was applied directly by injection under the skin above the fracture site. This volume 439 contained either a low dose of 0.2 μg hPTH 28–48 (Sigma-Aldrich Corporation, St. Louis, Missouri, USA), a high dose of 1.0 μg hPTH 28–48, 1.0 μg rat PTH 1–34 (Sigma-Aldrich Corporation, St. Louis, Missouri, USA), or vehicle (0.4% BSA in 0.01 mol/l acetic acid). hIL-6 (Sigma-Aldrich Corporation, St. Louis, Missouri, USA) was locally applied in the same conditions solely or in combination with it soluble receptor (hIL-6sR; Sigma-Aldrich Corporation, St. Louis, Missouri, USA) on days 7, 9, and 11 post-fracture. The following concentrations were applied: 8, 20, and 40 ng/0.2 ml of IL-6 and 20, 50, and 100 ng/0.2 ml of IL-6sR, respectively. Rats were euthenized by a lethal intraperitoneal injection of sodium phenobarbital on day 8, 14, or 6 weeks post-fracture. Processing for histology and histomorphometry Fractured left tibiae were removed by dislocation from the knee and ankle joints, and following removal of the pins were stripped of skin and carefully cleaned from surrounding soft tissue, keeping the periosteum intact as possible. Those that were removed from rats killed after 8 and 14 days were immersed in neutral buffered formalin solution for 48 h. Tibiae were then decalcified in 10% EDTA (in 0.1M Tris buffer) solution for 8 weeks. Following decalcification the tibiae were embedded in paraffin. Six micrometer thick sections were stained with Alcian blue (pH 2.5) and counterstained with H&E. Fig. 3. Representative sections of 14 days old callusses following injections on days 7, 9 and 11 of vehicle (A), 40 ng IL-6 (B), 8 ng IL-6 + 20 ng IL-6sR (C), 20 ng IL-6 + 50 ng IL-6sR (D), and 40 ng IL-6 + 100 ng IL-6sR (E). No differences were observed between vehicle-treated (A), IL-6 alone (B), and low doses of IL-6 + IL-6sR (C). Both combinations with the higher doses showed marked modulation of cartilage remodeling into bone (D&E). Alcian-blue-H&E staining. 440 N. Rozen et al. / Bone 41 (2007) 437–445 Sections that displayed a fracture gap between 4 cortices were chosen for analysis. Four Alcian blue-H&E-stained sections from each callus, approximately 50–100 μm apart, were analyzed morphometrically using an Olympus™ Image Analysis System (Cue-2 morphometry software of Galai Corporation, Migdal Haemek, Israel). Using a grid frame and tracing device, the surface area of 9 squares (3×3) of the grid in ×10 magnification (300 μm2) in 4 specific locations under the periosteum and adjacent to the cortices at the fracture gap (Fig. 1) was measured and surface areas occupied by fibrous tissue, cartilage, and cancellous bone were analyzed. Mean values ±SEM of areas of each type of tissue were calculated separately. Tibia volume measurement Before being exposed to the demineralization procedure and further processing for the histological observations, the 14 days treated tibiae from all experimental groups were subjected to calluses measurements by the principle of Archimedes. The left cleaned tibiae, which contained the fracture callus and periosteum and the corresponding right, similarly cleaned but not fractured tibiae, were immersed in 5 ml of distilled water in a 10 ml grading cylinder. The volume created by the immersed bones was measured and the additional volume, calculated by reducing the volume of the non-fractured from the fractured bone of the same rat represents callus volume. Mechanical testing Six weeks post-fracture calluses were immediately frozen in liquid nitrogen and kept frozen in −70 °C until the day of the mechanical testing. The night before testing, they were left to thaw in a refrigerator. The next day, all soft tissue was carefully removed. The mechanical strength of the healing fractures was measured by a 3-point bending procedure that was applied using a materials testing machine (Instron 1195, Instron Corporation, Canton, Massachusetts, USA). A small chamber containing three bars was tailor made for this purpose. The fractured bone was placed on two rounded bars (spaced 15 mm apart) with the fracture line between the bars. Deflection was performed by lowering another bar onto the fracture line using a constant speed of 1 mm/min. All bones were placed in the same position so that the force was applied from the anterior to the posterior surface of the bone—convex side was under pressure of the descending bar. Transducers recorded load and deflection continuously. The signal was fed to an x–y recorder, and the load–deflection curves obtained were read by a graphic tablet into a PC computer using Analog to Digital card (National Semiconductor, Santa Clara, California, USA). The parameters calculated were: maximum load to failure, absorptive energy, and stroke at breaking point. The stroke (mm) was defined as the length of deflection at maximum loading. Results were normalized and calculated with respect to the 2 diameters in the anterior-posterior and lateral directions of the tibia. analyzed by quantitative histomorphometry. Measurements of surface areas occupied by cartilage, intramembranous bone, and fibrous tissue revealed that by 8 days post-fracture (Fig. 2, upper panel) all 3 parameters increased when compared with vehicletreated fractures, but only cartilage and fibrous tissue increments caused by the local application of high dose PTH 28–48 were statistically significant: 2.8 (P b 0.02) and 1.7 (P b 0.02) -folds, respectively. By 14 days post-fracture (Fig. 2, lower panel), the increased cartilage surface area was maintained, but only the higher dose was statistically significant (1.8-folds, P b 0.02). As a consequence of these results, the dose of 1.0 μg was chosen for future experiments. IL-6 and IL-6 + IL-6sR dose response On days 7, 9, and 11 post-fracture 3 doses of IL-6 (8, 20 and 40 ng/0.2 ml) were injected solely or in combination with IL6sR (20, 50, and 100 ng/0.2 ml, respectively) and compared with vehicle-injected rats on the same days. All rats were sacrificed 14 days post-fracture, and their tibiae were processed for histological evaluation. All 3 doses of IL-6 tested showed a similar histological picture, namely quite a lot of cartilage Statistical analysis Histomorphometric and callus volume values are expressed as mean ± SEM. Statistical analysis of data was carried out using One-way Analysis of Variance (ANOVA), using t-test for means comparison between treated and control groups. Data were summed and calculated using SPSS statistics program. Results were regarded as significant at P b 0.05. Results Establishment of an effective dose of PTH 28–48 by differential histomorphometric analysis Fractures were implemented in the left tibiae of rats using the “Rozen Device”. On days 4, 5, and 6 post-fracture, all rats were injected locally, into the hematoma with vehicle, a low (0.2 μg; n = 10), and a high (1.0 μg; n = 10) dose of PTH 28–48. Five randomly selected rats from each group were sacrificed 8 days post-fracture and the rest on the 14th day. Paraffin sections were Fig. 4. Tibia volume measurements of 14 days old fractured and unfractured bones treated with vehicle (control), 40 ng IL-6+ 100 ng IL-6sR, 1.0 μg PTH 1–34, 1.0 μg PTH 28–48 and combinations. Results are means± SEM of 5 bones per group. *P b 0.0005—left (fractured) vs. right (unfractured); +P b 0.01—left (fractured) of an experimental rat vs. left (fractured) of vehicle treated control rat #P b 0.02—right (unfractured) of an experimental rat vs. right (unfractured) of vehicle treated control rat. N. Rozen et al. / Bone 41 (2007) 437–445 441 Fig. 5. Representative sections of 14 days old callusses: A—vehicle; B—increased amounts of cartilage following treatment with 1.0 μg PTH 28–48; C—mostly intramembranous bone in fractures that received the combination of 1.0 μg PTH 28–48 with 40 ng IL-6 + 100 ng IL-6sR. ca—cartilage, ct—cortex, eb—endosteal bone, pib—periosteal intramembranous bone, rf—resorption front. Alcian-blue-H&E staining. surrounding the fractured cortices, similar to vehicle-treated fractures (Figs. 3A and B). A gradual shift from cartilage to intramembranous bone was observed when IL-6 was combined with its soluble receptor. Although the combination with the low doses still resembled the vehicle-treated or IL-6 alone (Fig. 3C), both higher doses combinations showed marked modulation of cartilage remodeling into bone (Figs. 3D and E). As a consequence of these results, the combination of 40 ng IL-6 was chosen to combine with 100 ng of its soluble receptor for future experiments. Fig. 6. X-rays radiographs of fractured tibiae taken 5 weeks post-fracture. A—control; B—1.0 μg PTH 28–48; C—1.0 μg PTH 1–34; D—40 ng IL-6 followed by 100 ng IL-6sR; E—1.0 μg PTH 28–48 + 40 ng IL-6 + 100 ng IL-6sR; F—1.0 μg PTH 1–34 + 40 ng followed by IL-6 + 100 ng IL-6sR. Arrow—site of fracture. 442 N. Rozen et al. / Bone 41 (2007) 437–445 but not when compared to vehicle-treated fractured bones (Fig. 4). However both PTH fragments when combined with the later application of IL-6 + IL-6sR brought about a significant increase in tibia volume when compared both to their own unfractured tibiae controls and to vehicle-treated fractures (Fig. 4). Interestingly, a systemic effect on the unfractured right tibiae was exerted by the sequential application of PTH 1–34 followed by IL-6 + IL-6sR (Fig. 4, upper panel). Calculating the callus volume by deducting the volume of the unfractured tibiae from the fractured tibiae it can be appreciated that when PTH 1–34 was combined with IL-6 + IL-6sR, the ▵ increased by about 2.4-folds when compared with vehicle-treated rats (Fig. 4, upper panel) and by 3-folds when PTH 28–48 was combined with IL-6 + IL-6sR (Fig. 4, lower panel), probably because the increased cartilage area that was stimulated by PTH 28–48 served as infrastructure for its remodeling into bone. Indeed, comparison of the histology of 14 days old callusses showed increased amounts of cartilage following treatment with PTH 28–48 (Fig. 5B) when compared with vehicle-treated (Fig. 5A) and mostly intramembranous bone in fractures that received the combination of PTH 28–48 with IL-6 + IL-6sR (Fig. 5C). We concluded that IL-6 + IL-6sR accelerated the remodeling of cartilage into bone. X-rays radiography Fig. 7. Mechanical resistance as expressed by power to failure measurements (MPa) of 6 weeks old fractured and unfractured bones treated with vehicle (control), 1.0 μg PTH 1–34, 1.0 μg PTH 28–48 and combinations. Results are means ± SEM of 5 bones per group. *P b 0.0001 and b0.03—left (fractured) vs. right (unfractured) for PTH 1–34 + IL-6 + IL-6sR and PTH 28–48 + IL-6 + IL-6sR, respectively. +P b 0.0001 and b0.005—left (fractured) of an experimental rat vs. left (fractured) of vehicle treated control rat for PTH 1–34 + IL-6 + IL-6sR and PTH 28–48 + IL-6 + IL-6sR, respectively. X-rays radiographs that were taken by 5 weeks post-fracture showed that treatment of fractures with either of the PTH fragments resulted in enlarged calluses around the fracture site, Sequential application of PTH fragments and IL-6 + IL-6sR Following the results obtained by application of the mid fragment of PTH, PTH 28–48, and by application of IL-6 + IL-6sR, we decided to evaluate the sequential application of these factors on the fracture healing process. In addition we analyzed whether the known accelerating effect of the amino terminal fragment of PTH, PTH 1–34, is further enhanced by sequential application of IL-6 + IL-6sR. In order to be able to compare between both PTH fragments we applied also PTH 1–34 in the dose of 1.0 μg. The various factors were applied at the same temporal conditions as described in the former experiments. Two parameters were chosen for evaluation: measurement of tibia volume by 14 days post-fracture and analyzing mechanical resistance at 6 weeks post-fracture. Tibia volume PTH 1–34 was the only factor that increased tibia volume significantly when applied alone to fractured bones, but only when compared to the unfractured bones of the rats in its group Fig. 8. Graphs of the application of strength from representative control and experimental fractured bones. N. Rozen et al. / Bone 41 (2007) 437–445 while only fractures treated by sequential applications of either of the PTH fragments with IL-6 + IL-6sR demonstrated full healing (Fig. 6). Mechanical testing Fig. 7 demonstrates that both PTH fragments, when applied during the early stages of the healing process did not confer more resistance to 6 weeks healing bones, as measured using the 3 point-bending technique. However, a combination of both PTH fragments with IL-6 and its soluble receptor increased dramatically the fractures' resistance, 200% (P b 0.05) when PTH28–48 was combined and 300% (P b 0.0001) when PTH1–34 was combined. Moreover the resistance of the broken bones of the combination groups was similarly increased when compared with their own right unfractured bones (P b 0.03 and P b 0.00001, respectively, Fig. 7). Bones treated with IL-6 + IL-6sR were not measurable as these bones where easily broken during handling. Analysis of representative bones is presented in Fig. 8. In this figure it can be seen that as we have shown in Fig. 7, the mechanical load needed for callus break in both PTH fragments treated fractures did not differ from those treated with vehicle. In contrast, combination of both PTH fragments with IL-6 and its soluble receptor increased the plasticity of the bones as expressed in the increased load to failure needed to break the bones, in their energy absorptive capacity and in lengthening of the displacement parameter. Discussion Our working hypothesis was that the fracture healing process could be accelerated not only by the use of growth factors known to be anabolic to cartilage and bone, but also by factors that affect the remodeling of cartilage to bone in particular and bone turnover in general [1]. Therefore we opted to apply anabolic and remodeling accelerating agents in sequence at particular time points of the chondrogenic and cartilage to bone remodeling phases (4–6 and 7–11 days post-fracture, respectively) of the regeneration process. Our concept was to increase cartilage that will serve as an infrastructure for the newly formed bone and then to accelerate its remodeling into bone by endochondral ossification. PTH 1–34 and PTH 28–48 were chosen to stimulate the chondrogenic phase as they are known to be growth factors to skeletal cells [15,16,31,32]. In addition, on the basis of our previous studies that demonstrated activation of osteoclasts in nude mice by IL-6 + IL-6sR, we have chosen to use this cytokine as a cartilage to bone remodeling accelerator during fracture repair [29]. Preliminary experiments were performed in order to determine the dosage of local application of IL-6 into the hematoma and whether the addition of IL-6sR in order to improve its availability will be beneficial. Our results (shown in Fig. 5) demonstrate that remodeling was enhanced only when the soluble receptor for IL-6 was combined to the cytokine. In effect, it is well documented that the number of IL-6 receptors might be a limiting factor for its biological effect and that IL-6sR is a key factor for the effects of IL-6 in bone [25–28]. As a result of these experiments we decided to inject 40 ng IL-6 + 100 ng of the soluble receptor. 443 While the effects of the active amino-terminal fragment PTH 1–34 on fracture repair have been extensively studied [8,9,33–36], no data are available concerning its synthetic mid-region fragment, PTH 28–48. During the early stage of fracture healing the callus develops in order to stabilize the fractured bone fragments so that cortical union might proceed. As the main goal for intervention is acceleration of the healing process, we analyzed the effects of the PTH 28–48 peptide on callus components following its local application during the early stages of fracture healing. Due to the short half-life of this PTH fragment, we presumed that systemic administration would have little effect and opted to apply the fragment locally and directly to the encapsulated hematoma at the fracture site. This approach has been adopted by several authors, especially for the application of recombinant proteins with or without carriers, in order to circumvent undesired side effects that might accompany a systemic administration [2,37–41]. Our working hypothesis was that PTH 28–48 might be effective as an accelerating agent for fracture healing, as it has been proved by our group and others to induce chondrogenesis in mice and rats and also to exert mitogenic stimulation for osteoblastic cell lines [15–20]. Histomorphometric analysis of low-dose (0.2 μg) and high-dose (1.0 μg) injections demonstrated that by 8 days postfracture only cartilage and fibrous tissue increased significantly. However, by 14 days post-fracture only the increased cartilage surface area was maintained and only by the higher dose. As a consequence of these results, the dose of 1.0 μg was chosen for the following experiments. In summary, PTH 28–48, when injected directly into the callus area, stimulated all the components of the healing process but the fibrous tissue (that includes mainly mesenchynal progenitors) filling the gap and cartilage components were most profoundly stimulated. We suggest that the main effect of PTH 28–48 is to stimulate the proliferation of progenitor cells and orient their differentiation towards chondrogenesis. Concomitantly, it should be emphasized that woven bone formation was not compromised and proceeded undisturbed. Contrary to most studies that followed long-term daily systemic applications of different growth factors, we chose short-term local applications in accordance with specific biological stages of the healing process [1]. Thus, PTH fragments were injected solely on days 4, 5, and 6 in order to stimulate chondrogenesis, whereas IL-6 + IL-6sR were applied on days 7, 9, and 11 in order to accelerate cartilage and woven bone remodeling. Our results demonstrated that although both PTH fragments when applied during the early stages of the healing process enhanced callus formation by 14 days, this increased callus formation was not translated into greater resistance of the healing bone as measured 6 weeks post-fracture. On the other hand, a combination of each of the PTH fragments with IL-6 + IL-6sR increased not only the callus volume and accelerated the healing process as demonstrated by radiography, but also modified dramatically the quality of the bone, as presented by an increase of 300% in mechanical resistance when PTH 1–34 was combined and of 200% when PTH 28–48 was combined, when compared with vehicle-treated fractured bone. Interestingly, the mechanical strength of the contralateral unfractured bones of both experimental groups in which the PTH fragments combined with IL-6 + IL-6sR were 444 N. Rozen et al. / Bone 41 (2007) 437–445 injected to the callus was reduced compared to control unfractured bones. The probable explanation is that there is a systemic effect of the locally injected agents that increases remodeling of intact bones resulting in higher porosity and fragility. Several other agents and growth factors have been tested for their ability to accelerate fracture healing. Amongst them are the proteins of the BMP and TGF-β family. Increased callus volume in rat fractures that were locally treated with either BMP-2 or TGFβ were reported to be associated with only moderate increases in strength and stiffness [37,40], while in other studies in rabbits and rats treated with BMP-2, a temporal enhancement in the healing time was noted that was also accompanied with marked improvement of biomechanical properties [42–44]. Local application of TGF-β as a coating in poly (D,L-lactide)-coated titanium Kirschner wires resulted in less cartilage in the periosteal callus and increased mineralization and biomechanical strength [45], and BMP-2 when applied in a calcium phosphate paste enhanced temporal bridging and mineralized callus [46], exemplifying that the mode of application and the dosage have fundamental effects on the variation in the results. Exploring TGF-β effects when directly applied to the fracture site resulted in conflicting results. So, for example, no increase in callus volume under unstable fixation conditions, while some increase under stable conditions was reported in one study [37], while increase in the strength of tibial fractures was reported by others [39]. Applying one injection of 100 μg bFGF directly to the fracture site in rats had similar effects as PTH 28–48, namely enlargement of the cartilaginous callus with no induction of rapid healing [47], while doubling the dose stimulated bone remodeling and increased bone mineral content in a tibial fracture model in beagle dogs [48]. Perhaps the most promising results were achieved by intermittent application of PTH 1–34 throughout the healing process, probably by enhancing both proliferation and differentiation of osteoprogenitor cells and by increasing bone mineral content and osteoclastogenesis [8,9]. Similarly, a PTHrP analog had effective effects on impaired bone healing in rabbits that were on corticosteroid therapy [13]. As approximately 5–10% of fractures are associated with delayed or non-union healing, it is obvious that the search for agents that will be able to promote the healing process will continue. As of now no one specific factor has yet proved to gain clinical use, and may be in the future combinations of several factors will have to be explored [2,49]. Adopting this attitude PTH fragments carry promise for the future especially if combined with a factor that will concomitantly enhance the remodeling of the stimulated cartilage into a well-mineralized bone tissue. In conclusion the present study demonstrated that sequential application of anabolic PTH fragments with the remodeling accelerator IL-6 that stimulates chondro/osteoclasts function [50–51], results in a synergistic fracture healing enhancement. Further studies are needed as to how to integrate these findings into a potential for treating and accelerating fractures in the clinical setting. Acknowledgments We are grateful to Prof. Elazar Gutmanes from the Faculty of Materials and Engineering of the Technion for helping with the mechanical tests. 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