Research ArticleTissue Engineering

A Tissue Engineering Solution for Segmental Defect Regeneration in Load-Bearing Long Bones

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Science Translational Medicine  04 Jul 2012:
Vol. 4, Issue 141, pp. 141ra93
DOI: 10.1126/scitranslmed.3003720

Abstract

The reconstruction of large defects (>10 mm) in humans usually relies on bone graft transplantation. Limiting factors include availability of graft material, comorbidity, and insufficient integration into the damaged bone. We compare the gold standard autograft with biodegradable composite scaffolds consisting of medical-grade polycaprolactone and tricalcium phosphate combined with autologous bone marrow–derived mesenchymal stem cells (MSCs) or recombinant human bone morphogenetic protein 7 (rhBMP-7). Critical-sized defects in sheep—a model closely resembling human bone formation and structure—were treated with autograft, rhBMP-7, or MSCs. Bridging was observed within 3 months for both the autograft and the rhBMP-7 treatment. After 12 months, biomechanical analysis and microcomputed tomography imaging showed significantly greater bone formation and superior strength for the biomaterial scaffolds loaded with rhBMP-7 compared to the autograft. Axial bone distribution was greater at the interfaces. With rhBMP-7, at 3 months, the radial bone distribution within the scaffolds was homogeneous. At 12 months, however, significantly more bone was found in the scaffold architecture, indicating bone remodeling. Scaffolds alone or with MSC inclusion did not induce levels of bone formation comparable to those of the autograft and rhBMP-7 groups. Applied clinically, this approach using rhBMP-7 could overcome autograft-associated limitations.

Introduction

In orthopedic and trauma surgery, extensive bone loss is associated with major technical and biological problems. Bone grafts used to treat bone defects have the desired osteoconductive and osteoinductive properties. These “autografts,” however, have limited availability and are often difficult to access, causing further pain and additional healing time for the patient. Bone graft harvest causes donor site morbidity and increases the risk of infection, whereas transplants may also integrate insufficiently and require additional surgeries.

In search of alternative therapeutic strategies, research is focusing on the concept of tissue engineering to facilitate bone regeneration. Successful translation of tissue engineering strategies to the field of oral and maxillofacial surgery has motivated multidisciplinary teams to extend these concepts to long bone regeneration (1, 2). Long bone defects, however, are characterized by complex conditions eventually leading to nonunion or delayed healing (3). With predominantly soft tissue encasing and growing into such defects, the type and amount of cells, growth factors, and mechanical support required to achieve vascularization and to stimulate bone formation remain to be determined. Investigating the influence of each of these factors relies on using models that closely resemble the complex interrelations of impaired healing. Most small- and large-animal models—especially models relying on immature animals, such as young sheep and dogs—lack relevance to humans. Thus, we have established an ovine, critical-sized, tibial, mid-diaphyseal, segmental bone defect model to characterize the regenerative potential of medical-grade polycaprolactone–tricalcium phosphate (mPCL-TCP) scaffolds.

Here, we use these scaffolds either alone or in combination with autologous mesenchymal stem cells (MSCs) or recombinant human bone morphogenetic protein 7 (rhBMP-7). The mPCL scaffolds have U.S. Food and Drug Administration (FDA) approval and have received Conformité Européenne marking for the reconstruction of craniofacial bone defects. The second-generation scaffold, namely, mPCL-TCP, is currently in the FDA preapproval stage. The aim of this present study was to assess their regenerative potential in conjunction with either MSCs or rhBMP-7 compared to autologous cancellous bone graft (ABG), which is the current clinical gold standard for humans. We study this in a biomechanically and biologically demanding setting, closely resembling clinically relevant large segmental tibial defects. The results move toward addressing the important clinical question of how to treat large segmental bone defects by demonstrating scaffold-mediated regeneration and improved bone function compared to ABG controls after 1 year.

Results

Sheep model characterization

Sixty-four sheep of progressed age were chosen as study subjects because of their reduced intrinsic regenerative potential and their similarities to human bone regarding remodeling, turnover, and secondary osteon formation (4). Mid-diaphyseal tibial defects of 3-cm length were created and stabilized. The defects were left untreated/empty or were reconstructed with ABG, mPCL-TCP scaffolds, mPCL-TCP scaffolds and rhBMP-7 (fig. S1), or MSCs seeded into mPCL-TCP scaffolds (Table 1) using autologous platelet-rich plasma (PRP) for cell delivery. The animals were not immobilized after surgery to create the defect, which was important to mimic the effects of a weight-bearing bone.

Table 1

Biomechanical outcomes and μCT results after 3 and 12 months. Owing to lack of efficacy at 3 months, the scaffold/MSC group was not tested out to 12 months. Data represent median values.

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All animals were in good health and survived the 12-month in vivo study. No postoperative infections, implant failures, or macroscopic signs of foreign body reaction to the scaffolds occurred (fig. S2, A to C). One of eight animals treated with mPCL-TCP was excluded from analyses owing to a bone fracture through one of the proximal screw holes. One of eight animals treated with scaffold/rhBMP-7 showed evidence of a small and localized area of granulocytic infiltrate around the remnants of the collagen carrier (fig. S2C).

Biomechanical testing of implant–bone scaffold constructs

To determine whether the selected method of defect fixation was suitable to protect scaffolds from excessive loads, before the transplantation study, we investigated the mechanical behavior of the fixation implant–bone scaffold construct on sheep cadaver tibiae. Biomechanical testing in vitro showed that, under an axial compression load of 500 N, the interfragmentary movement (IFM) in the defect containing a scaffold was 0.27 mm, giving an interfragmentary strain (IFS = IFM/gap size) of less than 1%. There was minimal difference in the IFM (0.21 mm) under compression when an empty defect (scaffold removed from defect) was tested. When subjected to torsion (7 N·m), the fixation implant–bone scaffold construct underwent a relative rotation of the bone fragments of 7.4°. Medial-lateral bending induced by an axial load of 100 N at an offset of 10 cm resulted in a shortening of the defect axially by 4.0 mm (IFS, 13%), with a bending angle of 1.9°.

Bone healing 3 months after scaffold implantation

X-ray analysis after 3 months (Supplementary Materials) confirmed the critical-sized nature of the defect, as shown by a union rate of 0% for the empty control defects (Fig. 1A). For the ABG and rhBMP-7 groups, all eight animals (100%) showed bone bridging the defect (Fig. 1, B and D), but only three of eight (37.5%) showed bridging in the scaffold-only and MSC groups (Fig. 1, C and E). In all groups, distinct bone formation along the fixation plate was observed, which is a phenomenon also observed in people.

Fig. 1

Biomechanical outcomes after 3 months. (A to E) Representative x-ray images showing an empty control defect (n = 8) (A), a defect reconstructed with cancellous bone graft from the iliac crest (n = 8) (B), a defect treated with the mPCL-TCP scaffold only (n = 8) (C), and defects augmented with scaffold + rhBMP-7 (n = 8) (D) or scaffold + MSCs (n = 8) (E). Scale bar, 1 cm. (F) Median BVs were determined by CT. (G) BV distribution along the z axis. The total length of the defect was divided into three parts of equal length (proximal, middle, and distal thirds). (H and I) Torsional moment (H) and torsional stiffness (I) measurements. Empty control defects (n = 8) were excluded because they were filled with soft tissue only. *P < 0.05, Bonferroni-Holm.

Computed tomography (CT) values of total bone volume (BV) in the defect area were significantly higher with rhBMP-7 (8.6 cm3) when compared to all other scaffold-based groups (Fig. 1F). BV distribution along the defect’s z axis showed a tendency toward more bone formation at the defect/bone interfaces (Fig. 1G). For the empty control defects, no biomechanical testing could be performed owing to a lack of bony bridging, leaving the defects filled with soft tissue only. Torsional stiffness values were significantly higher for the scaffold/rhBMP-7 group (at two concentrations of BMP: 1.75 and 3.5 mg) when compared to the mPCL-TCP scaffold–only group at 3 months (Fig. 1, H and I). No significant difference in torsional moment or torsional stiffness was found between the ABG and the scaffold/rhBMP-7 groups, indicating that BMP-7 can induce bone of similar mechanical properties to ABG-mediated bone (Fig. 1, H and I). However, a significant difference in torsional stiffness was determined for ABG and the scaffold/rhBMP-7 groups when compared to scaffold/MSC (Fig. 1I), suggesting that MSCs alone are not able to regenerate bone as well as BMP-7.

The calculated BVs, BV distribution, and mechanical properties correlate well with macroscopic findings in micro-CT (μCT) reconstructions and histological sections in Fig. 2, fig. S3, and the corresponding animated three-dimensional (3D) μCT reconstructions of a representative sample of the control defects and the ABG, scaffold-only, and scaffold/rhBMP-7 groups (movies S1, S3, S4, and S6).

Fig. 2

Histological and imaging analyses of bone defects after 3 and 12 months. Images are representative of n = 3 to 8 samples; additional images are given in fig. S3. The empty defect was not studied at 12 months to avoid implant failures. The scaffold/MSC group was not studied at 12 months because it did not regenerate bone as well as ABG or the scaffold/rhBMP-7 combination. 3D μCT reconstructions and a frontal section of one representative sample per group, next to Safranin Orange/von Kossa–stained histology sections (orientation: top, proximal; left, medial).

Bone healing 12 months after treatment

Bone formation along the fixation plate—a common phenomenon in the clinic—and signs of cortex resorption in the proximity of the defect as indicated by a decreasing cortical density were observed in all animals (n = 23) (Fig. 2). 3D μCT reconstructions showed only partial defect bridging with the scaffold only (n = 7) (Fig. 2). In the ABG and scaffold/rhBMP-7 groups (n = 8 each), signs of bone remodeling were evident after 12 months, such as restored long bone morphology characterized by dense cortical bone and a marrow cavity composed of cancellous bone. Corresponding 3D animations of one sample of the ABG, scaffold-only, and scaffold/rhBMP-7 groups can be viewed in movies S2, S5, and S7. The amount of newly formed bone within each group varied considerably, as demonstrated in histological sections stained with Safranin Orange/von Kossa (fig. S4). No signs of scaffold degradation were evident (figs. S3 and S4).

Compared to the other treatments, overall mechanical strength (torsional moment) and torsional stiffness after 12 months were significantly higher when defects were augmented with the scaffold containing rhBMP-7 (Fig. 3, A and B, and Table 1). Improvements in strength and stiffness increased significantly over time for the ABG and scaffold/rhBMP-7 groups. For the scaffold-only group, torsional moment values increased minimally, but significantly, from 3 to 12 months, whereas torsional stiffness showed no significant change.

Fig. 3

Biomechanical testing and μCT results after 3 and 12 months. (A and B) Torsional moment (A) and torsional stiffness (B) represent biomechanics of empty defects (n = 8), ABG (n = 8), scaffold only (n = 7), and scaffold/rhBMP-7 (n = 8) over time. Data are median values normalized as a percentage against corresponding contralateral tibiae. Error bars are minimum and maximum values. (C) BVs were calculated as absolute values. (D) Median Jz values. All parameters were normalized as a percentage against corresponding contralateral tibiae. Error bars are minimum and maximum values. (E) Median TMD was normalized as a percentage against corresponding contralateral tibiae. Error bars are minimum and maximum values. *P < 0.05, Bonferroni-Holm.

At 12 months, BV and polar moment of inertia (Jz) remained significantly lower in the scaffold-only group compared to the scaffold/rhBMP-7 group (Fig. 3, C and D). When the scaffold-only group was compared to the ABG group, no difference was seen for BV (Fig. 3C). At 12 months, the scaffold/rhBMP-7–treated group exhibited higher BV and Jz values than the ABG group, suggesting that after 12 months, bone healing observed with scaffold/rhBMP-7 was superior compared to the gold standard autograft. For all three treatment groups, BV and Jz increased significantly over time (Fig. 3, C and D).

Last, the mineralization of ABG- and scaffold/rhBMP-7–treated defects increased between months 3 and 12, whereas no significant changes were observed for the scaffold-only group (Fig. 3E).

BV distribution after 12 months

BV distribution was determined using μCT in both the axial and the radial bone (Fig. 4A). Axial BV distribution was assessed in empty defects as well as in defects treated with ABG, scaffold only, or scaffold/rhBMP-7 by dividing the total length of the defect into three parts of equal length. In all treatment groups, there was a nonsignificant tendency toward greater bone formation in the proximal defect one-third, which is better vascularized, and more bone within the regions adjacent to the interfaces compared to the middle one-third, suggesting that defect regeneration is initiated by bone ingrowth at the defect regions proximate to the intact bone and subsequently advances toward the middle one-third (Fig. 4B).

Fig. 4

μCT analysis of axial and radial bone distribution after 3 and 12 months. (A) The definition of the VOI is depicted in the 2D longitudinal and transversal μCT tomograms. To describe BV distribution along the z axis, we divided the total defect length into three equal thirds (proximal, middle, and distal). The scaffold’s inner duct, its wall, and the periphery characterized radial bone distribution. (B) Axial bone formation in the distal, middle, and proximal thirds in control animals (empty defect, n = 8), ABG-treated animals (n = 8), and animals that received either scaffold only (n = 7 to 8) or scaffold/rhBMP-7 (n = 8). Data are medians ± minimum and maximum values. (C) Absolute values of radial bone formation in the three regions of interest in animals that received either scaffold only (n = 7 to 8) or scaffold/rhBMP-7 (n = 8). Data are medians ± minimum and maximum values. (D) Median radial BV in the scaffold’s inner duct and its wall was normalized as a percentage against the total VOI. Error bars are minimum and maximum values. *P < 0.05, Mann-Whitney U test, Bonferroni-Holm.

We assessed radial bone distribution in scaffold-only– and scaffold/rhBMP-7–treated animals, looking at the inner duct, the wall, and the periphery. At 3 and 12 months, the amount of new bone formed in the periphery in both groups was comparable to within the scaffold wall and inner duct (Fig. 4C). Radial bone distribution per unit volume of scaffold wall and inner duct was homogeneous in the scaffold-only group after both 3 and 12 months (Fig. 4D). With the addition of rhBMP-7 to the scaffold, there was a trend toward greater bone formation in the inner duct after 3 months, showing that BMP-7 locally increases bone formation mainly restricted to its site of application. At 12 months, however, significantly more bone was evident within the scaffold wall, indicating that BMP-7 drives bone remodeling (Fig. 4D) and the restoration of the tubular long bone morphology.

Bone tissue histology after 12 months

The morphology of the newly formed bone with scaffold/rhBMP-7 was investigated on histology sections stained with Movat’s pentachrome. Figure 5A shows the interface of old cortical bone and fibrolamellar bone with disorganized collagen fibers, which is characteristic for mammals when fast bone growth is required. At higher magnification, the vascularized, maturing bone tissue was observed to contain mineralized bone matrix, unmineralized osteoid, and mature osteocytes embedded in lacunae (Fig. 5B). The osteoid was located on the interface of mineralized bone and fibrous tissue (Fig. 5, C and D) and lined by bone-synthesizing osteoblasts and bone-resorbing osteoclasts. Blood vessels were embedded in soft tissue (Fig. 5, E and F).

Fig. 5

Morphology of newly formed bone in scaffold/rhBMP-7–treated animals. (A to F) Movat’s pentachrome stain. The histology sections show the interface of cortical bone and newly formed bone (A) and a higher-magnification image of the new bone (B). New bone could also be visualized around a scaffold strut with unmineralized osteoid (red) (C). Osteoblasts, osteoclasts, and mature osteocytes were embedded in lacunae (D), and blood vessels were embedded in soft tissue (light blue) (E and F). (G to I) BSE microscopy images of a transversal section of a contralateral tibia. These images illustrate the plexiform bone morphology characteristic of ovine bone comprising a combination of woven and lamellar bones within which vascular plexuses are sandwiched (G). Secondary osteon formation (yellow arrowheads) is seen in the vicinity of the marrow cavity (H and I).

Backscattered electron (BSE) imaging was used to characterize bone morphology of contralateral tibiae (Supplementary Materials). BSE imaging illustrates the largely plexiform bone morphology characteristic of ovine bone comprising a combination of woven and lamellar bones within which vascular plexuses are sandwiched (Fig. 5G). In the vicinity of the marrow cavity, secondary osteon formation was observed (Fig. 5, H and I, arrowheads). Notably, secondary, osteonal remodeling in sheep normally does not take place until an average age of 7 to 9 years (5).

Discussion

Segmental bone defects mostly occur at the tibial diaphysis because the tibia is the most commonly fractured long bone (up to 60% of segmental defects) (6). The overall rate of nonunion is as high as 21% (7, 8). Impaired functionality is a major clinical problem because the tibia is a weight-bearing bone and tibial shaft fractures often occur in young and active adults. We present a long-term (12-month) preclinical study in a segmental tibial animal model (sheep) that compares autograft treatment with the regenerative potential of composite scaffolds (9, 10) combined with autologous MSCs or rhBMP-7. The study demonstrates that critically sized segmental defects in long bones (tibiae) can be regenerated by recruitment and stimulation of endogenous cells by a scaffold/rhBMP-7 construct. X-ray, CT scans, μCT scans, biomechanical testing, and histology demonstrated successful repair of vascularized bone.

Sixty-four sheep of progressed age were chosen as study subjects for their reduced intrinsic regenerative potential and their close analogies with human bone regarding remodeling, turnover, and secondary osteon formation (4). Consequently, autograft augmented bone has had lower torsional moments and stiffnesses (11.19 and 19.32% of values, respectively, obtained for the contralateral limb) 3 months after surgery when compared to available literature that reports values of 40 to 50% for both torsional strength and stiffness (11, 12).

The composite scaffold materials applied have previously been shown to facilitate bone regeneration in craniofacial and spinal fusion–related defect sites in both pigs and humans, in which the scaffolds were implanted adjacent to large volumes of host bone (13, 14). However, with that implantation scenario, the mPCL-TCP scaffolds failed to induce defect consolidation. In our study, we have further compromised the intrinsic regenerative potential by thorough resection of the periosteum enveloping the tibial vascular bundle. The cambial layer of periosteum hosts mesenchymal progenitor cells and therefore provides a high regenerative potential (15). Nevertheless, the scaffold was able to provoke considerable bone ingrowth across the defect, which was not observed in empty control defects.

The mPCL-TCP used to fabricate the scaffolds has mechanical properties that allow for direct weight bearing, which was important because the animals were not immobilized after surgery. In vitro studies have shown that the material can be degraded by hydrolysis of the PCL ester linkages at physiological pH and temperature (16). As demonstrated in rabbits, degradation in vivo usually takes 2 to 3 years (9, 17), which is sufficient for complete bone healing in humans. Here, we confirmed by in vitro testing that the method of plate fixation was sufficient to protect the scaffolds from excessive loads during postoperative animal activity.

To increase the scaffolds’ osteogenic potential, we combined them with MSCs, which are able to differentiate into bone (18), and BMPs, which are well known to heal bone (19). rhBMP-7, in particular, stimulates bone healing by actively recruiting endogenous stem cells (20). Its beneficial effect on long bone healing in humans was demonstrated in various clinical trials, which have provided supportive evidence for the use of rhBMP-7 in the treatment of open or distal tibial fractures, tibial nonunions, and atrophic long bone nonunions (19, 21, 22). Successful union requires rhBMP-7 to be retained at the surgical site long enough to achieve osteoinduction (23). Under our study conditions, scaffold/MSC transplantation did not regenerate bone as well as ABG or the scaffold/rhBMP-7 combination after 3 months; reasons include the uncertainty about required cell numbers, the best state of differentiation, the time of transplantation, and the apoptosis of large numbers of cells after transplantation (24). Nevertheless, we believe that the local BMP delivery from the scaffolds induced a general increase in bone formation along with more bone deposition in the periphery of the defect, which brought about superior mechanical properties after 12 months, without the assistance of MSCs.

BMP-7 acts predominately in the early stages of fracture healing (25). We observed that rhBMP-7 improved and accelerated endochondral bone formation, vascularization, and the induction of osteoclast differentiation, thus accelerating the defect healing and the subsequent callus maturation. However, within the rhBMP-7 group, we observed a larger variation in bone formation. This could result from individual differences in the local mechanical environment, local pH, composition and size of the defect hematoma, surgical technique, release kinetics, and the concentration of local connective tissue progenitor cells or degree of vascularization (26, 27).

Our scaffolds did not degrade in the 12-month period. Formerly, it was advocated that scaffolds should degrade and vanish as the tissue is growing (28). Yet, tissue ingrowth does not equate to tissue maturation and remodeling, and a defect filled with immature tissue cannot be considered regenerated. Consequently, many previous scaffold-based strategies have failed, because the scaffold degradation occurred at a higher rate than tissue remodeling and/or maturation (29). The homogeneous distribution of mature tissue, however, ultimately leads to the desired mechanical stability. In our study, we observed no significant difference in bone formation between the proximal, the middle, and the distal thirds of the defects in any of the treatment groups. However, a tendency toward more bone formation in the proximal defect portions was observed. This could be explained by decreasing soft tissue coverage and vascularization from proximal defect regions to distal parts and an impaired blood supply to the distal end caused by the ostectomy. The phenomenon of reduced blood supply after osteotomy has previously been shown in adult rabbits (30).

Here, we did not examine the dose-dependent effects of rhBMP-7, which will be important for tailoring the scaffold-based concept to human use. All experimental studies using rhBMP-7 as a bone growth–stimulating adjuvant illustrate a dose-dependent effect and a species-dependent response variation (26, 31). The applied dosages range from 0.01 mg/ml in rats to 0.4 mg/ml in rabbits to more than 1.5 mg/ml in nonhuman primates. Different anatomical sites require different therapeutic doses depending on the degree of vascularization, defect size, and number of resident responding cells. On the basis of literature studies (32), we applied 3.5 mg of rhBMP-7 (0.5 to 0.6 mg/ml). Notably, the recommended dose of rhBMP-7 for recalcitrant long bone nonunions in humans remains 7 mg regardless of defect volume and shape (33).

We have not yet characterized the mechanism underlying host cell recruitment, diffusion, and histogenesis. It is known that BMPs stimulate osteoblasts to produce angiogenic factors, such as vascular endothelial growth factor and fibroblast growth factor (34), which results in recruitment and activation of endothelial cells. Direct effects of BMPs on endothelial cells include stimulation of migration, proliferation, and tube formation. Together, these effects of BMPs lead to increased neovascularization (35). We have previously shown in sheep that the mPCL-TCP scaffolds function as guiding substrates to the tissue formation process in healing bone defects (36) by enabling the arrangement of a structured fibrous tissue, which acted as a secondary supporting network for cells. Mineralization then initiated along the fibrous network, resulting in bone formation (36). If that process is further stimulated by a potent growth factor, such as BMP-7, the bone regeneration can theoretically be guided over larger distances, as shown in the present study in long bones.

The translation of tissue engineering concepts from bench to bedside is a challenging, expensive, and time-consuming process (3). Here, we describe our group’s approach to translate bone engineering from bench to bedside. We have established reliable methods for scaffold design and fabrication. The scaffolds’ mechanical, biochemical, and degradation characteristics have been investigated, and subsequent cell culture studies have confirmed their biocompatibility facilitating cell attachment, growth, and proliferation. Heterotopic and orthotopic proof-of-concept studies in small animals (3740) showed that our mPCL-TCP scaffolds do not evoke extensive inflammatory responses, are sufficiently strong, integrate well with the host tissue, allow vascularization, degrade slowly, and exhibit osteoconductive properties. We were able to increase their osteoinductivity by combination with rhBMP-7. Consequently, we now present the validation of the efficiency and efficacy of an mPCL-TCP scaffold/rhBMP-7 construct to regenerate a high-load–bearing, segmental, critical-sized bone defect in a large animal. The presented study outcomes, however, show that specific issues remain to be addressed before moving into people. These relate to growth factor immobilization on and release from the scaffolds, dosage, and the observed variation in bone formation within treatment groups.

Materials and Methods

MSC isolation and expansion

Under general anesthesia, bone marrow aspirates (5 to 10 ml) were obtained from the iliac crest. Total bone marrow cells (5 × 106 to 15 × 106 cells/ml) were plated at a density of 10 × 106 to 20 × 106 cells/cm2 in complete medium consisting of low-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 μg/ml). Cells were subsequently plated at a density of 103 cells/cm2. We previously demonstrated that these cells express the respective phenotypic profile characteristic of different mesenchymal cell populations and show a multilineage differentiation potential (38). Two weeks before implantation, the medium was changed to an osteogenic medium [DMEM, 10% FBS, penicillin (100 U/ml), streptomycin (100 μg/ml), β-glycerophosphate (10 μl/ml), ascorbic acid (1 μl/ml), and dexamethasone (1 μl/ml)] to induce osteogenic differentiation.

Preparation of autologous PRP

To prepare the PRP, we collected 80 ml of blood from the jugular vein and transferred it to 3.5-ml monovettes supplemented with sodium citrate (3.8%) at a ratio of 9:1 (41). The citrated blood was centrifuged for 20 min at 2400 rpm. Subsequently, the plasma supernatant was transferred to a 50-ml falcon tube and platelets were pelleted for 10 min at 3600 rpm (42). The pellet was resuspended in 2 ml of plasma, and the platelets were counted. By dilution with plasma, platelet concentrations were adjusted to 1 × 104/μl.

Scaffold fabrication and preparation

Cylindrical scaffolds of mPCL [number-average molecular weight (Mn), 80 kD; 1.145 g/cm3; Sigma-Aldrich] incorporating 20% β-TCP microparticles (Sigma-Aldrich; outer diameter, 20 mm; height, 30 mm; inner diameter, 8 mm) were fabricated by fused deposition modeling (Osteopore International). Scaffolds were pretreated with 1 M NaOH for 6 hours to render the scaffolds more hydrophilic and were sterilized. Filaments of about 300 μm in diameter were deposited after a 0/90° pattern with a separation of about 1200 μm, resulting in a scaffold with 70% porosity and fully interconnected pores. The scaffolds were characterized by an elastic modulus of 22.2 MPa.

The rhBMP-7 (Olympus Biotech Corporation) formulation consisted of 3.5 mg of rhBMP-7 formulated with 1 g of purified bovine type 1 collagen carrier. The product was reconstituted with 3 ml of saline to form a paste, which was then transferred to the inner duct of the scaffold and the contact interfaces between bone and scaffold (fig. S1).

MSC seeding was achieved by suspending 35 × 106 ovine MSCs in 250 μl of basal medium [low-glucose DMEM, 10% FBS, penicillin (100 U/ml), and streptomycin (100 μg/ml)]. The suspended cells were added to 1 ml of PRP and then seeded onto the sterilized scaffolds. PRP was activated with thrombin, which led to clotting and maintained the cells within the polymerized clot within the scaffold (5 U/ml). The scaffolds were placed in an incubator (37°C, 5% CO2) for 1 hour before transplantation.

Scaffold construct biomechanical testing

To investigate the mechanical behavior of the fixation implant–bone scaffold construct, we performed biomechanical testing in vitro. A fixation plate (Dynamic Compression Plate, Synthes) was affixed to a cylindrical bone analog cut to a length of 240 mm (fiber-filled epoxy cylinder with a 20-mm outer diameter and a 3-mm wall thickness; Pacific Research Laboratories). A 3-cm defect was created in the bone analog, and an mPCL-TCP scaffold was inserted into the defect. The ends of the construct were embedded in Paladur (Heraeus Kulzer), and the construct was mounted in a biaxial material testing machine (Instron 8874, Instron) with a custom-made jig. The test area was enclosed and heated to 37°C with a patient heater. The specimen was subjected to five cycles with the last cycle used for analysis. The custom-made jig was used to simulate three load cases: confined axial compression (500 N), axial torsion (7 N·m), and medial-lateral bending (10 N·m). To determine the IFMs at the center of the defect, we affixed optical marker rigid bodies (Orthopaedic Research Pins, Northern Digital Inc.) to the proximal and distal fragments of the bone analog. The displacements of the rigid bodies were captured by means of a motion capture system (Optotrak Certus, Northern Digital Inc.), and the IFMs were calculated with matrix algebra. The test was then repeated with the scaffold removed from the construct.

Segmental bone defect model

The study was approved by the animal ethics committee of the Queensland University of Technology (Brisbane, Australia; approval number: 700000915, 900000906). Under general anesthesia, 64 Merino sheep (average weight, 42.5 ± 3.7 kg; age, 6 to 7 years) underwent surgery to remove a 3-cm-long segment of the mid-diaphyseal tibia. The remaining bone was stabilized with a modified 10-hole Dynamic Compression Plate (Synthes) (43). Care was taken to remove the periosteum within the defect and 1 cm proximally and distally of the osteotomy lines. ABG was harvested in the form of cancellous particles from the left iliac crest of each animal. Experimental groups included (i) untreated control defects (n = 8), (ii) defects augmented with ABG (n = 16), (iii) defects reconstructed with mPCL-TCP scaffolds (n = 16), (iv) defects reconstructed with mPCL-TCP scaffolds and 3.5 mg of rhBMP-7 (n = 16), or (v) defects repaired with mPCL-TCP scaffolds seeded with MSCs (n = 8). Animals were allowed unrestricted weight bearing after recovery from anesthesia. Eight animals of each group were held for 3 months, whereas the remaining animals were held for a total period of 12 months (groups ii, iii, and iv). The 3-month end point was chosen because a study period of 12 weeks has previously been described as a suitable interval to allow detection of differences in bone repair (12). The late end point of 12 months was selected to enable the evaluation of long-term effects of the transplanted bone substitutes on bone regeneration and remodeling. Histological, x-ray, and CT analyses were performed upon sacrifice at both 3- and 12-month time points (Supplementary Materials).

Biomechanical testing of the operated limbs

After sacrifice, the operated tibiae were disarticulated. Both tibial ends were embedded in 80 ml of Paladur (Heraeus Kulzer) and mounted in a biaxial testing machine (Instron 8874). A torsion test was conducted under angular displacement control at an angular velocity of 0.5 °/s and a constant compressive preload of 0.05 kN until the first signs of fracture occurred. The process was stopped instantly to ensure structural integrity of the bones. The contralateral tibia was used as a paired reference from each animal. The torsional moment and torsional stiffness values were calculated from the slope of the torque-angular displacement curves and normalized against the values of the contralateral tibiae. Thereafter, specimens were fixed in 10% neutral buffered formalin solution before further processing.

Microcomputed tomography

A Viva40 μCT (Scanco Medical AG) was used to image and quantify the newly formed mineralized tissue after sacrifice at 3 and 12 months. Specimens were placed in a custom-made tube and scanned at a voltage of 70 kilovolt peak (kVp), a current of 114 μA, and a voxel size of 19 μm. The analyzed volume of interest (VOI) included the defect region only. Acquired images were segmented with a Gaussian filter of a width of 0.8 and support of 1.0. A threshold of 210 (519.2 mg of hydroxyapatite per cubic centimeter) was chosen for the defects, and a threshold of 230 (591.6 mg/cm3) was chosen for the contralateral tibiae. These were determined by visually evaluating 20 random tomograms per specimen of four samples per group, selecting a global threshold best depicting the morphology of mineralized tissue and excluding scaffold and soft tissue (44).

Within the VOI, the following parameters were determined: BV, axial BV distribution, radial bone distribution, Jz, and tissue mineral density (TMD). The Jz was calculated according to Jz = Ixx + Iyy, where Ixx and Iyy are the area moments of inertia about the other two mutually perpendicular axes.

Axial BV distribution was assessed by dividing the total length of the defect into three parts of equal length. The radial bone distribution was described by three VOIs: scaffold inner duct (VOIinner_duct), scaffold wall (VOIscaffold − VOIinner_duct), and scaffold periphery (VOItotal − VOIscaffold).

Statistical analysis

Statistical analysis was carried out with a two-tailed Mann-Whitney U test (SPSS 18.0, SPSS Inc.), and P values were adjusted according to Bonferroni-Holm. The presented box plots represent median, first quartile, and third quartile. The error bars indicate maximum and minimum values.

Supplementary Materials

www.sciencetranslationalmedicine.org/cgi/content/full/4/141/141ra93/DC1

Methods

Fig. S1. Scaffold preparation and transplantation.

Fig. S2. White blood cell infiltrate 12 months after reconstruction with scaffold/rhBMP-7.

Fig. S3. Histology of bone defects after 3 and 12 months.

Fig. S4. Images of histological sections stained with Safranin Orange/von Kossa.

Movie S1. ABG, 3 months.

Movie S2. ABG, 12 months.

Movie S3. Empty, 3 months.

Movie S4. Scaffold only, 3 months.

Movie S5. Scaffold only, 12 months.

Movie S6. Scaffold and rhBMP-7, 3 months.

Movie S7. Scaffold and rhBMP-7, 12 months.

References and Notes

  1. Acknowledgments: We thank the staff at the Queensland University of Technology Medical Engineering Research Facility for veterinary assistance and administrative and technical support. We also thank M. Princ and M. Thiele for their technical assistance. Funding: Centro de Estudios e Investigaciones Técnicas, Universidad de Navarra, Spain (A.C.); the Australian Research Council and the Wesley Research Foundation, Brisbane; and the Berlin-Brandenburg Center for Regenerative Therapies. Author contributions: J.C.R. was responsible for technical design, developing the animal model, performing all animal surgeries, x-rays, clinical CT scans, and mechanical testing, and drafting the paper. A.C. performed the μCT analyses, 3D animations, histological analyses, and BSE imaging and participated in the writing of the paper. P.K. developed the method of quantitative clinical CT analysis. D.R.E. helped to develop the method of quantitative clinical CT analysis and assisted with the mechanical testing, clinical CT scans, and data interpretation. A.B. and S.S. participated in animal surgery and assisted with x-rays, clinical CT scans, and cell culture. H.S. and M.A.W. assisted with the histological analysis. M.A.W. further contributed to drafting the paper. M.M. assisted with μCT scans. D.W.H. initiated and directed the project. D.W.H., M.A.S., and G.N.D. conceived and helped design the experiments, oversaw the collection of results and data interpretation, and finalized the paper. Competing interests: The authors declare that they have no competing interests.
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