AMERI CAN JOURNAL OF OT OLA RYNGOLOGY–H E A D A N D N EC K ME D IC IN E AN D S U RG ER Y 36 ( 20 1 5 ) 1 –6
Available online at www.sciencedirect.com
ScienceDirect www.elsevier.com/locate/amjoto
Original contributions
Biomimetic scaffolds facilitate healing of critical-sized segmental mandibular defects☆,☆☆,★ Matthew K. Lee, MD a , Adam S. DeConde, MD a , Min Lee, PhD b, c , Christopher M. Walthers, MS c , Ali R. Sepahdari, MD d , David Elashoff, PhD e , Tristan Grogan, MS e , Olga Bezouglaia f , Sotirios Tetradis, DDS, PhD f, g , Maie St. John, MD PhD a, g,⁎, Tara Aghaloo, DDS, MD, PhD f, g,⁎⁎ a
Department of Head and Neck Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA, United States Division of Advanced Prosthodontics, Biomaterials, and Hospital Dentistry, UCLA School of Dentistry, Los Angeles, CA, United States c Department of Bioengineering, UCLA School of Engineering and Applied Sciences, Los Angeles, CA, United States d Department of Radiology, David Geffen School of Medicine at UCLA, Los Angeles, CA, United States e Department of Medicine Statistics Core, David Geffen School of Medicine at UCLA, Los Angeles, CA, United States f Division of Diagnostic and Surgical Sciences, UCLA School of Dentistry, Los Angeles, CA, United States g Jonsson Comprehensive Cancer Center, David Geffen School of Medicine at UCLA, Los Angeles, CA, United States b
ARTI CLE I NFO
A BS TRACT
Article history:
Objective: To investigate the efficacy of biomimetic PLGA scaffolds, alone and in
Received 10 March 2014
combination with bone morphogenic protein (BMP-2) and adipose-derived stem cells (ASCs), to heal a critical-sized segmental mandibular defect in a rat model. Study design: Prospective animal study. Methods: ASCs were isolated and cultured from the inguinal fat of Lewis rat pups. Using three-dimensional printing, PLGA scaffolds were fabricated and impregnated with BMP-2 and/or ASCs. Critical-sized 5-mm segmental mandibular defects were created in adult Lewis rats and implanted with (1) blank PLGA scaffolds, (2) PLGA scaffolds with ASCs, (3) PLGA scaffolds with BMP, or (4) PLGA scaffolds with BMP and ASCs. Animals were sacrificed at 12 weeks. Bone regeneration was assessed using microCT, and graded on a semiquantitative bone formation and bone union scale. Results: Twenty-eight rats underwent creation of segmental mandibular defects with implantation of scaffolds. Nine rats suffered complications and were excluded from analysis, leaving 19 animals for inclusion in the study. MicroCT analysis demonstrated no bridging of the segmental bony defect in rats implanted with blank scaffolds (median bone union score = 0). Rats implanted with scaffolds containing BMP-2 (median bone union = 2.0), ASCs (median bone union = 1.5), and combination of BMP and ASCs (median bone
☆
Conflict of Interest: None. Financial Disclosures: None. ★ Funding for this research was provided by: The American Academy of Otolaryngology–Head & Neck Surgery Foundation Resident Research Grant, Award ID 274718. ⁎ Correspondence to: M. St. John, Department of Head and Neck Surgery, David Geffen School of Medicine at UCLA, CHS 62-132, 10833 Le Conte Ave, Los Angeles, CA 90095-1624, United States. Tel.: + 310 206-6766. ⁎⁎ Correspondence to: T. Aghaloo, Division of Diagnostic and Surgical Sciences, UCLA School of Dentistry, CHS 53-076 10833 LeConte Ave, Los Angeles, CA 90095-1668, United States. Tel.: + 310 794-7070. E-mail addresses: [email protected] (M. St. John), [email protected] (T. Aghaloo).
☆☆
http://dx.doi.org/10.1016/j.amjoto.2014.06.007 0196-0709/© 2015 Elsevier Inc. All rights reserved.
2
AMERI CAN JOURNAL OF OT OLAR YNGOLOGY–H E A D A N D NE CK ME D IC IN E A ND S U RG ER Y 36 ( 20 1 5 ) 1 –6
union = 1.0) demonstrated healing of critical-sized segmental mandibular defects. Bone regeneration was most robust in the BMP-2 treated scaffolds. Conclusions: The current study utilizes a novel animal model to study the efficacy of biomimetic scaffolds carrying osteogenic factors to induce healing of a critical-sized segmental mandibular defect. Level of evidence: N/A, Basic Science Animal Research. © 2015 Elsevier Inc. All rights reserved.
1.
Introduction
Segmental mandibular defects may result secondary to the treatment of a variety of pathologies, including benign and malignant tumors involving the mandible, radiation or druginduced osteonecrosis, or as a complication of traumatic injury [1,2]. Reconstruction of the mandible is required in order to restore occlusion, oral competence, and facial contour, and thereby obviate the detrimental quality of life consequences of segmental mandibular defects [3,4]. Currently, microvascular free tissue transfer is considered the gold standard for oromandibular reconstruction [3,5–7]. However, free flap surgery is lengthy and is associated with perioperative complication rates affecting up to 30%–40% of patients [8]. Tissue engineering has emerged as a promising new area of investigation exploring alternative methods for mandibular reconstruction. In both in vitro and in vivo studies, poly(lacticco-glycolic acid), or PLGA, has been utilized as a biomimetic scaffold for delivery of osteogenic growth factors, such as bone morphogenetic protein (BMP), or as a delivery system for adiposederived stem cells (multipotent stem cells with the potential for ostegenic differentiation) [9,10]. Animal studies have been performed utilizing a variety of animal models, including canine, primate, and rabbit [11–14]. These large-scale animal models provide a useful model of the volume and load-bearing conditions of the defects seen in humans, but are also comparatively large and expensive. Recently, our lab described a novel small animal model (rat) for the study of segmental mandibular defects [1] and established 5-mm as the critical-sized defect, i.e. defect size that will not heal spontaneously [15]. The purpose of the current study is to investigate methods for regeneration of critical-sized segmental mandibular defects with the use of tissue engineering techniques. Specifically, the role of PLGA scaffolds loaded with BMP-2 and adipose-derived stem cells (ASCs) is explored.
2.
Materials and methods
2.1.
Animals
All animal care and use complied with institutional regulations established and approved by the Animal Research Committee at the University of California, Los Angeles, an AALAC-accredited facility. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animal of the National Institutes of Health. Adult Lewis rats (Charles River Laboratories, Wilmington, MA) were maintained in a temperature-regulated environ-
ment (24 °C) on a 12-h light/dark cycle. Animals were housed one pair per cage, with soft bedding and a microisolator cover. All animals were initially kept on a soft (Nutra-Gel, Bio-Serv, Frenchtown, NJ) diet for one week following surgery, after which they were advanced to a regular diet (Bacon Softies, Bio-Serv, Frenchtown, NJ). Weights were recorded weekly to ensure that all animals were maintaining an adequate level of nutrition (defined as < 10% loss of body weight). Animals were divided into four experimental groups: (1) blank scaffolds, (2) scaffolds containing BMP-2, (3) scaffolds containing ASCs, and (4) scaffolds containing a combination of BMP-2 and ASCs. In each of these four groups, 5-mm segmental defects were created. The resulting defect was then rigidly fixed using 1-mm titanium miniplates, and the defect implanted with the appropriate scaffold. All animals were sacrificed 12 weeks following surgery unless there was development of perioperative complications (infection, plate extrusion, etc.). In these cases, animals were euthanized at the time of complication discovery. Lewis rat pups served as a source of adipose-derived stem cells, and were sacrificed on postnatal days 3 to 5 to harvest inguinal fat pads. Inbred Lewis rats were selected to allow for implantation of ASCs from pups to adult without necessitating immunosuppression and its associated adverse effects.
2.2.
Isolation of adipose-derived stem cells
Lewis rat pups were sacrificed on postnatal days 3 to 5. Inguinal fat pads were harvested, and adipose-derived stem cells were isolated and cultured as follows. Inguinal fat pads were washed three times with 1% penicillin/streptomycin solution (Gibco® PenStrep, Life Technologies, Grand Island, NY) and treated with 0.075% collagenase (type I; SigmaAldrich, St. Louis, MO) in PBS for 30 min at 37 °C under gentle agitation. Enzymatic digestion was inactivated by addition of DMEM/10% fetal bovine serum (FBS). The cell suspension was centrifuged for 10 min, and the resulting pellet resuspended in standard cell medium (DMEM/10% FBS, Gibco® Life Technologies, Grand Island, NY) and filtered through a 100-μm mesh filter to remove residual debris. Cells were then plated in cell medium and cultured until confluence was achieved.
2.3.
Preparation of PLGA scaffolds
Apatite-coated PLGA scaffolds were created from 85:15 poly (lactic-co-glycolic acid) (inherent viscosity = 0.61 dL/g, Birmingham Polymers) through a solvent casting/particulate leaching process, as has been previously described [10,16]. In brief, PLGA/chloroform solutions were mixed with
AMERI CAN JOURNAL OF OT OLA RYNGOLOGY–H E A D A N D N EC K ME D IC IN E AN D S U RG ER Y 36 ( 20 1 5 ) 1 –6
3
200–300 μm diameter sucrose to obtain 92% porosity (volume fraction), and compressed into 5 × 10 × 2-mm Teflon molds. Scaffolds were then freeze-dried overnight. Sucrose was removed by submerging scaffolds in 3 washes of distilled H2O, freeing the scaffold from the Teflon mold. After leaching, all scaffolds were immersed in 50%, 60% and 70% ethanol for 30 min each, followed by rinses of distilled H2O. Apatite coating was achieved by incubating scaffolds in simulated body fluid (SBF). All scaffolds were then allowed to dry under a laminar flow hood.
2.4.
Seeding of scaffolds with ASCs and BMP
Selected scaffolds were then loaded with BMP-2 or ASCs. For BMP-2-loaded scaffolds, 0.5 μg of rhBMP-2 (R&D Systems, Minneapolis, MN) was adsorbed by dropping the solution onto scaffolds over a period of 20 min. Further lyophilization was then accomplished in a freeze drier. For ASC-seeded scaffolds, scaffolds were first immersed in cell medium for 1 h prior to seeding, after which excess cell medium was aspirated. ASCs which had been grown on a culturing plate were then digested with 0.05% trypsin for 5 min under gentle agitation. Enzymatic digestion was halted by addition of cell medium followed by 3 washes with sterile PBS. The resulting cell suspension was then centrifuged, and the resulting cell pellet resuspended to a density of 1 × 107 cells per 1 mL. Each scaffold was then seeded with 1,000,000 ASCs (100 μL) in a 24-well plate for 1.5 h. Following seeding, additional cell medium was added and scaffolds were incubated overnight 1 day prior to implantation.
2.5.
Creation of critical-sized defect
Animals underwent general anesthesia with inhalational isoflurane. Animals were then shaved, prepped, and draped in sterile fashion. A #15 blade was used to create an incision overlying and paralleling the left mandible. This was then deepened down through subcutaneous tissues until the inferior border of the mandible was identified. The mandible was further exposed by dividing the pterygomasseteric sling using electrocautery, and the musculature bluntly elevated off the lingual and buccal surfaces of the mandible in a supraperiosteal plane. A 5-mm segmental defect was measured. Two 1-mm miniplates (one superior and one inferior) were then placed over the mandible and drill holes were pre-drilled prior to creation of the bony cuts, with 2 points of fixation on either side of the planned defect for the inferior plate, and 1 point of fixation on either side of the defect for the superior plate. A 1-mm high-speed cutting burr (set at 3000 RPM) was used to drill the defect under copious irrigation. Hemostasis was achieved with electrocautery and the wound irrigated free of bone dust. The appropriate scaffold was then placed into the mandibular defect (Fig. 1), and the pterygomasseteric sling reapproximated with absorbable sutures. The skin was closed with nylon sutures in a simple running fashion. Rats were then allowed to recover from anesthesia and transferred to the vivarium for postoperative monitoring. Postoperatively, all animals received analgesia with subcutaneous injections of buprenorphine (0.1 mg/kg) for 72-h
Fig. 1 – Scaffold placed within segmental mandibular defect.
postoperatively. All animals also received trimethoprim– sulfamethoxazole in the water supply for one week following the operation as prophylaxis against infection.
2.6.
MicroCT analysis
Animals were sacrificed twelve weeks following surgery. Left hemi-mandibles were harvested and fixed in 10% formalin for 48 h. Imaging was performed using high-resolution microCT (μCT40; Scanco USA, Inc., Southeastern, PA). MicroCT data were collected at 50 kVp and 160 μA, and images were reconstructed in three dimensions using Dolphin 3D imaging software (Dolphin Imaging & Management Solutions, Chatsworth, CA). 3D surface reconstructions of the mandible, including the regions of interest (ROI) were made using the entire volumetric data set. Scatter artifact from the titanium microplates precluded quantitative analysis of the scans as artifact cannot be reliably distinguished from newly formed bone by current software. Therefore, the examiners, blinded to the identity of the specimens, were allowed to utilize the software's dynamic segmentation function to create the most realistic appearance of the mandible with minimal loss of unwounded cortical bone due to thin structures and minimal superimposition of artifacts and soft tissue. A previously described semi-quantitative scale of bone union and bone formation was then utilized to evaluate healing of the segmental defect [17]. Three clinicians each independently reviewed all images and recorded their scores according to the previously described scale.
2.7.
Statistical analysis
To assess the pairwise agreement of raters we computed the percentage of agreement and the κ statistic. Each rat was measured twice for bone formation and union. The median score of those 2 measurements was used for the analysis. To evaluate overall differences between the four groups (control, BMP, ASCs, and combination BMP and ASCs) a Kruskal–Wallis
4
AMERI CAN JOURNAL OF OT OLAR YNGOLOGY–H E A D A N D NE CK ME D IC IN E A ND S U RG ER Y 36 ( 20 1 5 ) 1 –6
test was carried out. If the Kruskal–Wallis test was statistically significant, pairwise differences between the three groups were tested with the Mann–Whitney U test. Comparisons between groups were deemed statistically significant at the α < 0.05 threshold. Statistical analyses and plots were carried out with R (Version 2.15.0) and SPSS (Version 19).
3.
Results
3.1.
Complications
Healing of critical-sized segmental mandibular defects
Bone union and bone formation scores were graded according to a previously described semi-quantitative scale [17] by three independent clinicians. Median bone union and bone formation scores are summarized in Table 1. The Kruskal–Wallis test demonstrated a statistically significant difference in the distribution of bone union scores between the four groups (p = 0.015). Subsequently, pairwise differences between the four groups were evaluated with the Mann–Whitney U test (Table 2). This demonstrated greater bone union in the BMP-treated group (p = 0.010) and the combination BMP/ASC-treated group (p = 0.014), when compared to the control group. The ASC-treated group demonstrated an overall higher median bone union score when compared to the control group, though this did not reach statistical significance (p = 0.131). Fig. 2 illustrates a microCT image of a healed segmental mandibular defect in the BMP-2 group. Kruskal–Wallis test demonstrated no significant difference in distribution of bone formation by group (p = 0.302).
3.3.
Group 1 Control Control Control
versus versus versus
Group 2
p Value
BMP-2 ASC BMP-2 + ASC
0.010 ⁎ 0.343 0.014 ⁎
⁎ Statistically significant at α < 0.05 threshold.
all raters (κ = 0.58 for bone union scores, κ = 0.70 for bone formation scores).
Twenty-eight animals were included in this study, 7 per each experimental group. Nine animals (32.1%) developed exposure of the titanium miniplates, requiring exclusion from the study. No other complications were noted. There were no intraoperative deaths. At the end of 12 weeks, there were no animals that suffered from critical loss of weight (defined as > 10% of initial body weight). A total of 19 animals were included in the final analysis.
3.2.
Table 2 – Pairwise differences of bone union scores.
Inter-observer reliability
Inter-observer variability was evaluated using the multirater κ statistic. This demonstrated substantial agreement between
Table 1 – Bone union and formation scores. Group
Bone union (median)
Bone formation (median)
Control BMP-2 ASC BMP-2 + ASC
0.0 2.0 0.5 1.0
0.5 2.0 1.5 1.0
BMP-2: Bone morphogenetic protein-2; ASC: Adipose-derived stem cells.
4.
Discussion
Microvascular free tissue transfer, specifically the free vascularized bone graft, has gained general acceptance as the preferred method for reconstruction of oromandibular defects. Free tissue transfer has been shown to possess excellent reliability and consistency as a reconstructive modality with success rates ranging from 96% to 99% [3–7]. Although an effective method of mandibular reconstruction, free flap surgery is not without its limitations. Prolonged operative times subject patients to significant medical risks, with perioperative complications reported as high as 30%–40% [8]. In elderly patients with multiple comorbid conditions, this risk can rise to greater than 60% [18]. Free flap reconstruction therefore contributes to the potential morbidity and overall complexity of surgical treatment for mandibular defects. Investigations into alternative methods of mandibular reconstruction are therefore warranted in an effort to decrease perioperative medical comorbidity. Various osteoinductive growth factor-based therapies have been developed in an attempt to find an effective and safer method of bone regeneration. Bone morphogenetic proteins (BMPs) are among the most potent osteoinductive factors available and have been extensively studied for the treatment of critical-sized bony defects [19,20]. BMP-2 is perhaps the most thoroughly investigated member of the BMP family, and is currently FDAapproved for bone healing in spinal fusion, maxillary sinus augmentation, and localized alveolar ridge defects [15]. Research into the osteogenic potential of cellular-based therapies has also recently been burgeoning, with significant efforts kindled by the discovery of adipose-derived stem cells (ASCs) [21]. ASCs readily differentiate down an osteogenic lineage, both in vitro and in vivo [22,23]. Recently, Parrilla et al. [24] describe their results using a critical-sized rat mandibular trephine defect model, showing successful bony regeneration and healing with implantation of ASCs. Given the ease of harvest and readily available autologous sources, ASCs have demonstrated alluring potential as a treatment option for regeneration of mandibular defects. In the current study we have employed a small-animal model for segmental mandibular defects, initially described by our lab [1]. Our goal was to investigate the potential of BMP2 and ASCs to heal an established critical-sized (5-mm) segmental mandibular defect in a novel small animal model. This model was selected for a number of compelling reasons. Small animal models provide numerous advantages when
AMERI CAN JOURNAL OF OT OLA RYNGOLOGY–H E A D A N D N EC K ME D IC IN E AN D S U RG ER Y 36 ( 20 1 5 ) 1 –6
5
Fig. 2 – Left: 3D reconstruction of healed defect. Blue lines indicate the span of the original segmental defect. Right: Axial cross section through the middle of a healed defect, demonstrating formation of trabecular bone.
compared to larger animals (i.e. primate, canine, rabbit) [11–14] specifically due to the reduced financial cost related to animal husbandry in addition to ethical implications. Also, when using small animal models, previous studies investigating mandibular regeneration have typically employed trephine or marginal mandibular defects [25–27]. While no perfect animal model exists to replicate the masticatory forces and volume considerations encountered in human mandibular defect, marginal and trephine defects (when compared to segmental models) provide a less accurate simulation of the defects most commonly encountered in humans following mandibular resection. In our previous investigations using this small animal model, we established 5-mm as the critical-sized segmental mandibular defect, i.e. defect size that will not heal spontaneously despite rigid internal fixation [15]. In the current study, we investigate the role of BMP-2 and ASCs to heal this critical-sized defect. PLGA scaffolds were engineered to the exact defect size and were used to deliver BMP-2, ASCs, or a combination of both factors. Given PLGA’s lack of inherent osteoinductive potential [28], blank PLGA scaffolds were unable to produce healing of segmental mandibular defects when implanted in isolation (all 4 animals demonstrated a bone union score of 0). PLGA scaffolds impregnated with BMP-2, however, demonstrated robust osteogenic potential, with all 5 of 5 animals demonstrating successful bony union (as defined by a radiographic evidence of a bone bridge spanning the defect, yielding a bone union score of at least 1, assessed at the 12 week time point). This also held true for the combination group (scaffolds containing both BMP and ASCs), with 5 of 6 animals demonstrating a defect-spanning bony bridge. When mean bone union scores for the BMP group and the combination BMP/ASC group were compared to controls, pairwise analysis demonstrated a statistically significant improvement in bone union scores (p = 0.010 and 0.014, respectively). Interestingly, scaffolds seeded with ASCs alone showed moderate osteogenic potential, with 2 of 4 animals having a minimum bone union score of 1 (indicating presence of an intact bony bridge in 50% of animals). However, when comparing mean and median bone union scores of the ASC
group to those of the controls, this difference trended toward but did not reach statistical significance by pairwise analysis (p = 0.131). Though clinically, ASCs do appear to have the ability to induce healing of a segmental mandibular defect, the statistical significance of our findings may have been limited by the small overall number of animals in each group, and specifically in the ASC group (which only had 4 animals, fewer than the other two experimental groups). Bone formation scores were found not to vary significantly between the treatment groups (p = 0.302). While all three treatment groups (BMP, ASC, and combination) yielded higher bone formation scores than the control group, animals with blank PLGA scaffolds still demonstrated some bony regrowth (mean bone formation score = 0.8). However, in animals implanted with blank scaffolds, this occurred in an unorganized fashion from the margins of the osteotomies, as evidenced by lack of formation of a defect-spanning bony bridge. By contrast, with use of BMP and ASCs, bone regrowth occurred primarily within the confines of the PLGA scaffolds. This suggests that the use of PGLA scaffolds impregnated with osteogenic factors facilitates both the degree and organization of bone regeneration, both critical elements for successful healing of a segmental mandibular defect. The findings presented here represent an important initial investigation into the study of osteogenic factors for regeneration of mandibular defects utilizing a small animal model. The current study demonstrates the robust bone regeneration potential of BMP-2 using a PLGA scaffold as a delivery vehicle. These findings corroborate those of previous studies employing large animal models, and provide validation for the smallanimal segmental mandibular defect currently utilized. The use of a reproducible, reliable small animal model is significant, as the lower cost of animal husbandry facilitates initial investigations into the bone regeneration potential of novel therapies.
5.
Conclusion
The current study utilizes a novel animal model to study the efficacy of biomimetic scaffolds loaded with various
6
AMERI CAN JOURNAL OF OT OLAR YNGOLOGY–H E A D A N D NE CK ME D IC IN E A ND S U RG ER Y 36 ( 20 1 5 ) 1 –6
osteogenic factors to potentiate bone regeneration and induce healing of an established critical-sized segmental mandibular defect. We find that these results represent a significant development in the field of small animal mandibular regeneration research. Using an affordable, reproducible small animal model that simulates segmental defects commonly encountered in humans, we have investigated the potential of various osteogenic factors to produce bony union within a segmental mandibular defect. This model can be similarly utilized in the future in animal research to investigate the role of new and potentially promising osteogenic agents.
REFERENCES
[1] Sidell DR, Aghaloo T, Tetradis S, et al. Composite mandibulectomy a novel animal model. Otolaryngol Head Neck Surg 2012;146:932–7. [2] Suh JD, Blackwell KE, Sercarz JA, et al. Disease relapse after segmental resection and free flap reconstruction for mandibular osteoradionecrosis. Otolaryngol Head Neck Surg 2010;142:586–91. [3] Blackwell KE. Unsurpassed reliability of free flaps for head and neck reconstruction. Arch Otolaryngol Head Neck Surg 1999;125:295–9. [4] Urken ML, Buchbinder D, Costantino PD, et al. Oromandibular reconstruction using microvascular composite flaps: report of 210 cases. Arch Otolaryngol Head Neck Surg 1998;124:46–55. [5] Hidalgo DA, Pusic AL. Free-flap mandibular reconstruction: a 10-year follow-up study. Plast Reconstr Surg 2002;110:438–49 [discussion 450]. [6] Wax MK, Rosenthal E. Etiology of late free flap failures occurring after hospital discharge. Laryngoscope 2007;117:1961–3. [7] Nuara MJ, Sauder CL, Alam DS. Prospective analysis of outcomes and complications of 300 consecutive microvascular reconstructions. Arch Facial Plast Surg 2009;11:235–9. [8] Suh JD, Sercarz JA, Abemayor E, et al. Analysis of outcome and complications in 400 cases of microvascular head and neck reconstruction. Arch Otolaryngol Head Neck Surg 2004;130:962–6. [9] Deconde AS, Sidell D, Bezouglaia O, Low K, Elashoff D, Grogan T, Tetradis S, Aghaloo T, St. John M. BMP-2 impregnated biomimetic scaffolds successfully induce bone healing in a marginal mandibular defect. Laryngoscope. in press. [10] Levi B, James AW, Nelson ER, et al. Human adipose derived stromal cells heal critical size mouse calvarial defects. PLoS One 2010;5:e11177. [11] Zwetyenga S, Catros A, Emparanza C, et al. Mandibular reconstruction using induced membranes with autologous cancellous bone graft and HA-BTCP: animal model study and preliminary results in patients. Int J Oral Maxillofac Surg 2009;38:1289–97. [12] He Y, Zhang Z, Han-Guang Z, et al. Experimental study on reconstruction of segmental mandible defects using tissue engineered bone combined bone marrow stromal cells with three dimensional tricalcium phosphate. J Craniofac Surg 2007;18:800–5.
[13] Elsalanty ME, Zakhary I, Akeel S, et al. Reconstruction of canine mandibular bone defects using a bone transport reconstruction plate. Ann Plast Surg 2009;63:441–8. [14] Seto I, Asahina I, Oda M, Enomoto S. Reconstruction of the primate mandible with a combination graft of recombinant human bone morphogenic protein-2 and bone marrow. J Oral Maxillofac Surg 2001;59:53–61. [15] Deconde AS, Lee MK, Sidell D, et al. Establishing the critical-sized defect in the rat segmental mandibulectomy animal model. Oral presentation at the AAO-HNSF Annual Meeting, September 2012; 2012. [16] Lee M, Chen TT, Iruela-Arispe ML, Wu BM, Dunn JCY. Modulation of protein delivery from modular polymer scaffolds. Biomaterials 2007;28:1862–70. [17] Johnson KD, Frierson KE, Keller TS, et al. Porous ceramics as bone graft substitutes in long bone defects: a biomechanical, histological, and radiographic analysis. J Orthop Res 1996;14:351–69. [18] Blackwell KE, Azizzadeh B, Ayala C, Rawnsley JD. Octogenarian free flap reconstruction: complications and cost of therapy. Otolaryngol Head Neck Surg 2002;126:301–6. [19] Kang Q, Sun MH, Cheng H, et al. Characterization of the distinct orthotopic bone-forming activity of 14 BMPs using recombinant adenovirus-mediated gene delivery. Gene Ther 2004;11:1312–20. [20] Govender S, Csimma C, Genant HK, Valentin-Opran A, Grp BS. Recombinant human bone morphogenetic protein-2 for treatment of open tibial fractures—a prospective, controlled, randomized study of four hundred and fifty patients. J Bone Joint Surg Am 2002;84A:2123–34. [21] Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 2001;7:211–28. [22] Jeon O, Rhie JW, Kwon I, Kim J, Kim B, Lee S. In vivo bone formation following transplantation of human adipose-derived stromal cells that are not differentiated osteogenically. Tissue Eng A 2008;14:1285–94. [23] Asti A, Gastaldi G, Dorati R, et al. Stem cells grown in osteogenic medium on PLGA, PLGA/HA, and titanium scaffolds for surgical applications. Bioinorg Chem Appl 2010:831031–43. [24] Parrilla C, Saulnier N, Bernardini C, et al. Undifferentiated human adipose tissue-derived stromal cells induce mandibular bone healing in rats. Arch Otolaryngol Head Neck Surg 2011;137:463–70. [25] Schliephake H, Weich HA, Dullin C, Gruber R, Frahse S. Mandibular bone repair by implantation of rhBMP-2 in a slow release carrier of polylactic acid—an experimental study in rats. Biomaterials 2008;29:103–10. [26] Issa JPM, do Nascimento C, Iyomasa MM, et al. Bone healing process in critical-sized defects by rhBMP-2 using poloxamer gel and collagen sponge as carriers. Micron 2008;39:17–24. [27] Arosarena OA, Collins WL. Bone regeneration in the rat mandible with bone morphogenetic protein-2: a comparison of two carriers. Otolaryngol Head Neck Surg 2005;132:592–7. [28] Kruger EA, Im D, Bischoff DS, et al. In vitro mineralization of human mesenchymal stem cells on three-dimensional type I collagen versus PLGA scaffolds: a comparative analysis. Plast Reconstr Surg 2011;127:2301–11.
Available online at www.sciencedirect.com
ScienceDirect www.elsevier.com/locate/amjoto
Original contributions
Biomimetic scaffolds facilitate healing of critical-sized segmental mandibular defects☆,☆☆,★ Matthew K. Lee, MD a , Adam S. DeConde, MD a , Min Lee, PhD b, c , Christopher M. Walthers, MS c , Ali R. Sepahdari, MD d , David Elashoff, PhD e , Tristan Grogan, MS e , Olga Bezouglaia f , Sotirios Tetradis, DDS, PhD f, g , Maie St. John, MD PhD a, g,⁎, Tara Aghaloo, DDS, MD, PhD f, g,⁎⁎ a
Department of Head and Neck Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA, United States Division of Advanced Prosthodontics, Biomaterials, and Hospital Dentistry, UCLA School of Dentistry, Los Angeles, CA, United States c Department of Bioengineering, UCLA School of Engineering and Applied Sciences, Los Angeles, CA, United States d Department of Radiology, David Geffen School of Medicine at UCLA, Los Angeles, CA, United States e Department of Medicine Statistics Core, David Geffen School of Medicine at UCLA, Los Angeles, CA, United States f Division of Diagnostic and Surgical Sciences, UCLA School of Dentistry, Los Angeles, CA, United States g Jonsson Comprehensive Cancer Center, David Geffen School of Medicine at UCLA, Los Angeles, CA, United States b
ARTI CLE I NFO
A BS TRACT
Article history:
Objective: To investigate the efficacy of biomimetic PLGA scaffolds, alone and in
Received 10 March 2014
combination with bone morphogenic protein (BMP-2) and adipose-derived stem cells (ASCs), to heal a critical-sized segmental mandibular defect in a rat model. Study design: Prospective animal study. Methods: ASCs were isolated and cultured from the inguinal fat of Lewis rat pups. Using three-dimensional printing, PLGA scaffolds were fabricated and impregnated with BMP-2 and/or ASCs. Critical-sized 5-mm segmental mandibular defects were created in adult Lewis rats and implanted with (1) blank PLGA scaffolds, (2) PLGA scaffolds with ASCs, (3) PLGA scaffolds with BMP, or (4) PLGA scaffolds with BMP and ASCs. Animals were sacrificed at 12 weeks. Bone regeneration was assessed using microCT, and graded on a semiquantitative bone formation and bone union scale. Results: Twenty-eight rats underwent creation of segmental mandibular defects with implantation of scaffolds. Nine rats suffered complications and were excluded from analysis, leaving 19 animals for inclusion in the study. MicroCT analysis demonstrated no bridging of the segmental bony defect in rats implanted with blank scaffolds (median bone union score = 0). Rats implanted with scaffolds containing BMP-2 (median bone union = 2.0), ASCs (median bone union = 1.5), and combination of BMP and ASCs (median bone
☆
Conflict of Interest: None. Financial Disclosures: None. ★ Funding for this research was provided by: The American Academy of Otolaryngology–Head & Neck Surgery Foundation Resident Research Grant, Award ID 274718. ⁎ Correspondence to: M. St. John, Department of Head and Neck Surgery, David Geffen School of Medicine at UCLA, CHS 62-132, 10833 Le Conte Ave, Los Angeles, CA 90095-1624, United States. Tel.: + 310 206-6766. ⁎⁎ Correspondence to: T. Aghaloo, Division of Diagnostic and Surgical Sciences, UCLA School of Dentistry, CHS 53-076 10833 LeConte Ave, Los Angeles, CA 90095-1668, United States. Tel.: + 310 794-7070. E-mail addresses: [email protected] (M. St. John), [email protected] (T. Aghaloo).
☆☆
http://dx.doi.org/10.1016/j.amjoto.2014.06.007 0196-0709/© 2015 Elsevier Inc. All rights reserved.
2
AMERI CAN JOURNAL OF OT OLAR YNGOLOGY–H E A D A N D NE CK ME D IC IN E A ND S U RG ER Y 36 ( 20 1 5 ) 1 –6
union = 1.0) demonstrated healing of critical-sized segmental mandibular defects. Bone regeneration was most robust in the BMP-2 treated scaffolds. Conclusions: The current study utilizes a novel animal model to study the efficacy of biomimetic scaffolds carrying osteogenic factors to induce healing of a critical-sized segmental mandibular defect. Level of evidence: N/A, Basic Science Animal Research. © 2015 Elsevier Inc. All rights reserved.
1.
Introduction
Segmental mandibular defects may result secondary to the treatment of a variety of pathologies, including benign and malignant tumors involving the mandible, radiation or druginduced osteonecrosis, or as a complication of traumatic injury [1,2]. Reconstruction of the mandible is required in order to restore occlusion, oral competence, and facial contour, and thereby obviate the detrimental quality of life consequences of segmental mandibular defects [3,4]. Currently, microvascular free tissue transfer is considered the gold standard for oromandibular reconstruction [3,5–7]. However, free flap surgery is lengthy and is associated with perioperative complication rates affecting up to 30%–40% of patients [8]. Tissue engineering has emerged as a promising new area of investigation exploring alternative methods for mandibular reconstruction. In both in vitro and in vivo studies, poly(lacticco-glycolic acid), or PLGA, has been utilized as a biomimetic scaffold for delivery of osteogenic growth factors, such as bone morphogenetic protein (BMP), or as a delivery system for adiposederived stem cells (multipotent stem cells with the potential for ostegenic differentiation) [9,10]. Animal studies have been performed utilizing a variety of animal models, including canine, primate, and rabbit [11–14]. These large-scale animal models provide a useful model of the volume and load-bearing conditions of the defects seen in humans, but are also comparatively large and expensive. Recently, our lab described a novel small animal model (rat) for the study of segmental mandibular defects [1] and established 5-mm as the critical-sized defect, i.e. defect size that will not heal spontaneously [15]. The purpose of the current study is to investigate methods for regeneration of critical-sized segmental mandibular defects with the use of tissue engineering techniques. Specifically, the role of PLGA scaffolds loaded with BMP-2 and adipose-derived stem cells (ASCs) is explored.
2.
Materials and methods
2.1.
Animals
All animal care and use complied with institutional regulations established and approved by the Animal Research Committee at the University of California, Los Angeles, an AALAC-accredited facility. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animal of the National Institutes of Health. Adult Lewis rats (Charles River Laboratories, Wilmington, MA) were maintained in a temperature-regulated environ-
ment (24 °C) on a 12-h light/dark cycle. Animals were housed one pair per cage, with soft bedding and a microisolator cover. All animals were initially kept on a soft (Nutra-Gel, Bio-Serv, Frenchtown, NJ) diet for one week following surgery, after which they were advanced to a regular diet (Bacon Softies, Bio-Serv, Frenchtown, NJ). Weights were recorded weekly to ensure that all animals were maintaining an adequate level of nutrition (defined as < 10% loss of body weight). Animals were divided into four experimental groups: (1) blank scaffolds, (2) scaffolds containing BMP-2, (3) scaffolds containing ASCs, and (4) scaffolds containing a combination of BMP-2 and ASCs. In each of these four groups, 5-mm segmental defects were created. The resulting defect was then rigidly fixed using 1-mm titanium miniplates, and the defect implanted with the appropriate scaffold. All animals were sacrificed 12 weeks following surgery unless there was development of perioperative complications (infection, plate extrusion, etc.). In these cases, animals were euthanized at the time of complication discovery. Lewis rat pups served as a source of adipose-derived stem cells, and were sacrificed on postnatal days 3 to 5 to harvest inguinal fat pads. Inbred Lewis rats were selected to allow for implantation of ASCs from pups to adult without necessitating immunosuppression and its associated adverse effects.
2.2.
Isolation of adipose-derived stem cells
Lewis rat pups were sacrificed on postnatal days 3 to 5. Inguinal fat pads were harvested, and adipose-derived stem cells were isolated and cultured as follows. Inguinal fat pads were washed three times with 1% penicillin/streptomycin solution (Gibco® PenStrep, Life Technologies, Grand Island, NY) and treated with 0.075% collagenase (type I; SigmaAldrich, St. Louis, MO) in PBS for 30 min at 37 °C under gentle agitation. Enzymatic digestion was inactivated by addition of DMEM/10% fetal bovine serum (FBS). The cell suspension was centrifuged for 10 min, and the resulting pellet resuspended in standard cell medium (DMEM/10% FBS, Gibco® Life Technologies, Grand Island, NY) and filtered through a 100-μm mesh filter to remove residual debris. Cells were then plated in cell medium and cultured until confluence was achieved.
2.3.
Preparation of PLGA scaffolds
Apatite-coated PLGA scaffolds were created from 85:15 poly (lactic-co-glycolic acid) (inherent viscosity = 0.61 dL/g, Birmingham Polymers) through a solvent casting/particulate leaching process, as has been previously described [10,16]. In brief, PLGA/chloroform solutions were mixed with
AMERI CAN JOURNAL OF OT OLA RYNGOLOGY–H E A D A N D N EC K ME D IC IN E AN D S U RG ER Y 36 ( 20 1 5 ) 1 –6
3
200–300 μm diameter sucrose to obtain 92% porosity (volume fraction), and compressed into 5 × 10 × 2-mm Teflon molds. Scaffolds were then freeze-dried overnight. Sucrose was removed by submerging scaffolds in 3 washes of distilled H2O, freeing the scaffold from the Teflon mold. After leaching, all scaffolds were immersed in 50%, 60% and 70% ethanol for 30 min each, followed by rinses of distilled H2O. Apatite coating was achieved by incubating scaffolds in simulated body fluid (SBF). All scaffolds were then allowed to dry under a laminar flow hood.
2.4.
Seeding of scaffolds with ASCs and BMP
Selected scaffolds were then loaded with BMP-2 or ASCs. For BMP-2-loaded scaffolds, 0.5 μg of rhBMP-2 (R&D Systems, Minneapolis, MN) was adsorbed by dropping the solution onto scaffolds over a period of 20 min. Further lyophilization was then accomplished in a freeze drier. For ASC-seeded scaffolds, scaffolds were first immersed in cell medium for 1 h prior to seeding, after which excess cell medium was aspirated. ASCs which had been grown on a culturing plate were then digested with 0.05% trypsin for 5 min under gentle agitation. Enzymatic digestion was halted by addition of cell medium followed by 3 washes with sterile PBS. The resulting cell suspension was then centrifuged, and the resulting cell pellet resuspended to a density of 1 × 107 cells per 1 mL. Each scaffold was then seeded with 1,000,000 ASCs (100 μL) in a 24-well plate for 1.5 h. Following seeding, additional cell medium was added and scaffolds were incubated overnight 1 day prior to implantation.
2.5.
Creation of critical-sized defect
Animals underwent general anesthesia with inhalational isoflurane. Animals were then shaved, prepped, and draped in sterile fashion. A #15 blade was used to create an incision overlying and paralleling the left mandible. This was then deepened down through subcutaneous tissues until the inferior border of the mandible was identified. The mandible was further exposed by dividing the pterygomasseteric sling using electrocautery, and the musculature bluntly elevated off the lingual and buccal surfaces of the mandible in a supraperiosteal plane. A 5-mm segmental defect was measured. Two 1-mm miniplates (one superior and one inferior) were then placed over the mandible and drill holes were pre-drilled prior to creation of the bony cuts, with 2 points of fixation on either side of the planned defect for the inferior plate, and 1 point of fixation on either side of the defect for the superior plate. A 1-mm high-speed cutting burr (set at 3000 RPM) was used to drill the defect under copious irrigation. Hemostasis was achieved with electrocautery and the wound irrigated free of bone dust. The appropriate scaffold was then placed into the mandibular defect (Fig. 1), and the pterygomasseteric sling reapproximated with absorbable sutures. The skin was closed with nylon sutures in a simple running fashion. Rats were then allowed to recover from anesthesia and transferred to the vivarium for postoperative monitoring. Postoperatively, all animals received analgesia with subcutaneous injections of buprenorphine (0.1 mg/kg) for 72-h
Fig. 1 – Scaffold placed within segmental mandibular defect.
postoperatively. All animals also received trimethoprim– sulfamethoxazole in the water supply for one week following the operation as prophylaxis against infection.
2.6.
MicroCT analysis
Animals were sacrificed twelve weeks following surgery. Left hemi-mandibles were harvested and fixed in 10% formalin for 48 h. Imaging was performed using high-resolution microCT (μCT40; Scanco USA, Inc., Southeastern, PA). MicroCT data were collected at 50 kVp and 160 μA, and images were reconstructed in three dimensions using Dolphin 3D imaging software (Dolphin Imaging & Management Solutions, Chatsworth, CA). 3D surface reconstructions of the mandible, including the regions of interest (ROI) were made using the entire volumetric data set. Scatter artifact from the titanium microplates precluded quantitative analysis of the scans as artifact cannot be reliably distinguished from newly formed bone by current software. Therefore, the examiners, blinded to the identity of the specimens, were allowed to utilize the software's dynamic segmentation function to create the most realistic appearance of the mandible with minimal loss of unwounded cortical bone due to thin structures and minimal superimposition of artifacts and soft tissue. A previously described semi-quantitative scale of bone union and bone formation was then utilized to evaluate healing of the segmental defect [17]. Three clinicians each independently reviewed all images and recorded their scores according to the previously described scale.
2.7.
Statistical analysis
To assess the pairwise agreement of raters we computed the percentage of agreement and the κ statistic. Each rat was measured twice for bone formation and union. The median score of those 2 measurements was used for the analysis. To evaluate overall differences between the four groups (control, BMP, ASCs, and combination BMP and ASCs) a Kruskal–Wallis
4
AMERI CAN JOURNAL OF OT OLAR YNGOLOGY–H E A D A N D NE CK ME D IC IN E A ND S U RG ER Y 36 ( 20 1 5 ) 1 –6
test was carried out. If the Kruskal–Wallis test was statistically significant, pairwise differences between the three groups were tested with the Mann–Whitney U test. Comparisons between groups were deemed statistically significant at the α < 0.05 threshold. Statistical analyses and plots were carried out with R (Version 2.15.0) and SPSS (Version 19).
3.
Results
3.1.
Complications
Healing of critical-sized segmental mandibular defects
Bone union and bone formation scores were graded according to a previously described semi-quantitative scale [17] by three independent clinicians. Median bone union and bone formation scores are summarized in Table 1. The Kruskal–Wallis test demonstrated a statistically significant difference in the distribution of bone union scores between the four groups (p = 0.015). Subsequently, pairwise differences between the four groups were evaluated with the Mann–Whitney U test (Table 2). This demonstrated greater bone union in the BMP-treated group (p = 0.010) and the combination BMP/ASC-treated group (p = 0.014), when compared to the control group. The ASC-treated group demonstrated an overall higher median bone union score when compared to the control group, though this did not reach statistical significance (p = 0.131). Fig. 2 illustrates a microCT image of a healed segmental mandibular defect in the BMP-2 group. Kruskal–Wallis test demonstrated no significant difference in distribution of bone formation by group (p = 0.302).
3.3.
Group 1 Control Control Control
versus versus versus
Group 2
p Value
BMP-2 ASC BMP-2 + ASC
0.010 ⁎ 0.343 0.014 ⁎
⁎ Statistically significant at α < 0.05 threshold.
all raters (κ = 0.58 for bone union scores, κ = 0.70 for bone formation scores).
Twenty-eight animals were included in this study, 7 per each experimental group. Nine animals (32.1%) developed exposure of the titanium miniplates, requiring exclusion from the study. No other complications were noted. There were no intraoperative deaths. At the end of 12 weeks, there were no animals that suffered from critical loss of weight (defined as > 10% of initial body weight). A total of 19 animals were included in the final analysis.
3.2.
Table 2 – Pairwise differences of bone union scores.
Inter-observer reliability
Inter-observer variability was evaluated using the multirater κ statistic. This demonstrated substantial agreement between
Table 1 – Bone union and formation scores. Group
Bone union (median)
Bone formation (median)
Control BMP-2 ASC BMP-2 + ASC
0.0 2.0 0.5 1.0
0.5 2.0 1.5 1.0
BMP-2: Bone morphogenetic protein-2; ASC: Adipose-derived stem cells.
4.
Discussion
Microvascular free tissue transfer, specifically the free vascularized bone graft, has gained general acceptance as the preferred method for reconstruction of oromandibular defects. Free tissue transfer has been shown to possess excellent reliability and consistency as a reconstructive modality with success rates ranging from 96% to 99% [3–7]. Although an effective method of mandibular reconstruction, free flap surgery is not without its limitations. Prolonged operative times subject patients to significant medical risks, with perioperative complications reported as high as 30%–40% [8]. In elderly patients with multiple comorbid conditions, this risk can rise to greater than 60% [18]. Free flap reconstruction therefore contributes to the potential morbidity and overall complexity of surgical treatment for mandibular defects. Investigations into alternative methods of mandibular reconstruction are therefore warranted in an effort to decrease perioperative medical comorbidity. Various osteoinductive growth factor-based therapies have been developed in an attempt to find an effective and safer method of bone regeneration. Bone morphogenetic proteins (BMPs) are among the most potent osteoinductive factors available and have been extensively studied for the treatment of critical-sized bony defects [19,20]. BMP-2 is perhaps the most thoroughly investigated member of the BMP family, and is currently FDAapproved for bone healing in spinal fusion, maxillary sinus augmentation, and localized alveolar ridge defects [15]. Research into the osteogenic potential of cellular-based therapies has also recently been burgeoning, with significant efforts kindled by the discovery of adipose-derived stem cells (ASCs) [21]. ASCs readily differentiate down an osteogenic lineage, both in vitro and in vivo [22,23]. Recently, Parrilla et al. [24] describe their results using a critical-sized rat mandibular trephine defect model, showing successful bony regeneration and healing with implantation of ASCs. Given the ease of harvest and readily available autologous sources, ASCs have demonstrated alluring potential as a treatment option for regeneration of mandibular defects. In the current study we have employed a small-animal model for segmental mandibular defects, initially described by our lab [1]. Our goal was to investigate the potential of BMP2 and ASCs to heal an established critical-sized (5-mm) segmental mandibular defect in a novel small animal model. This model was selected for a number of compelling reasons. Small animal models provide numerous advantages when
AMERI CAN JOURNAL OF OT OLA RYNGOLOGY–H E A D A N D N EC K ME D IC IN E AN D S U RG ER Y 36 ( 20 1 5 ) 1 –6
5
Fig. 2 – Left: 3D reconstruction of healed defect. Blue lines indicate the span of the original segmental defect. Right: Axial cross section through the middle of a healed defect, demonstrating formation of trabecular bone.
compared to larger animals (i.e. primate, canine, rabbit) [11–14] specifically due to the reduced financial cost related to animal husbandry in addition to ethical implications. Also, when using small animal models, previous studies investigating mandibular regeneration have typically employed trephine or marginal mandibular defects [25–27]. While no perfect animal model exists to replicate the masticatory forces and volume considerations encountered in human mandibular defect, marginal and trephine defects (when compared to segmental models) provide a less accurate simulation of the defects most commonly encountered in humans following mandibular resection. In our previous investigations using this small animal model, we established 5-mm as the critical-sized segmental mandibular defect, i.e. defect size that will not heal spontaneously despite rigid internal fixation [15]. In the current study, we investigate the role of BMP-2 and ASCs to heal this critical-sized defect. PLGA scaffolds were engineered to the exact defect size and were used to deliver BMP-2, ASCs, or a combination of both factors. Given PLGA’s lack of inherent osteoinductive potential [28], blank PLGA scaffolds were unable to produce healing of segmental mandibular defects when implanted in isolation (all 4 animals demonstrated a bone union score of 0). PLGA scaffolds impregnated with BMP-2, however, demonstrated robust osteogenic potential, with all 5 of 5 animals demonstrating successful bony union (as defined by a radiographic evidence of a bone bridge spanning the defect, yielding a bone union score of at least 1, assessed at the 12 week time point). This also held true for the combination group (scaffolds containing both BMP and ASCs), with 5 of 6 animals demonstrating a defect-spanning bony bridge. When mean bone union scores for the BMP group and the combination BMP/ASC group were compared to controls, pairwise analysis demonstrated a statistically significant improvement in bone union scores (p = 0.010 and 0.014, respectively). Interestingly, scaffolds seeded with ASCs alone showed moderate osteogenic potential, with 2 of 4 animals having a minimum bone union score of 1 (indicating presence of an intact bony bridge in 50% of animals). However, when comparing mean and median bone union scores of the ASC
group to those of the controls, this difference trended toward but did not reach statistical significance by pairwise analysis (p = 0.131). Though clinically, ASCs do appear to have the ability to induce healing of a segmental mandibular defect, the statistical significance of our findings may have been limited by the small overall number of animals in each group, and specifically in the ASC group (which only had 4 animals, fewer than the other two experimental groups). Bone formation scores were found not to vary significantly between the treatment groups (p = 0.302). While all three treatment groups (BMP, ASC, and combination) yielded higher bone formation scores than the control group, animals with blank PLGA scaffolds still demonstrated some bony regrowth (mean bone formation score = 0.8). However, in animals implanted with blank scaffolds, this occurred in an unorganized fashion from the margins of the osteotomies, as evidenced by lack of formation of a defect-spanning bony bridge. By contrast, with use of BMP and ASCs, bone regrowth occurred primarily within the confines of the PLGA scaffolds. This suggests that the use of PGLA scaffolds impregnated with osteogenic factors facilitates both the degree and organization of bone regeneration, both critical elements for successful healing of a segmental mandibular defect. The findings presented here represent an important initial investigation into the study of osteogenic factors for regeneration of mandibular defects utilizing a small animal model. The current study demonstrates the robust bone regeneration potential of BMP-2 using a PLGA scaffold as a delivery vehicle. These findings corroborate those of previous studies employing large animal models, and provide validation for the smallanimal segmental mandibular defect currently utilized. The use of a reproducible, reliable small animal model is significant, as the lower cost of animal husbandry facilitates initial investigations into the bone regeneration potential of novel therapies.
5.
Conclusion
The current study utilizes a novel animal model to study the efficacy of biomimetic scaffolds loaded with various
6
AMERI CAN JOURNAL OF OT OLAR YNGOLOGY–H E A D A N D NE CK ME D IC IN E A ND S U RG ER Y 36 ( 20 1 5 ) 1 –6
osteogenic factors to potentiate bone regeneration and induce healing of an established critical-sized segmental mandibular defect. We find that these results represent a significant development in the field of small animal mandibular regeneration research. Using an affordable, reproducible small animal model that simulates segmental defects commonly encountered in humans, we have investigated the potential of various osteogenic factors to produce bony union within a segmental mandibular defect. This model can be similarly utilized in the future in animal research to investigate the role of new and potentially promising osteogenic agents.
REFERENCES
[1] Sidell DR, Aghaloo T, Tetradis S, et al. Composite mandibulectomy a novel animal model. Otolaryngol Head Neck Surg 2012;146:932–7. [2] Suh JD, Blackwell KE, Sercarz JA, et al. Disease relapse after segmental resection and free flap reconstruction for mandibular osteoradionecrosis. Otolaryngol Head Neck Surg 2010;142:586–91. [3] Blackwell KE. Unsurpassed reliability of free flaps for head and neck reconstruction. Arch Otolaryngol Head Neck Surg 1999;125:295–9. [4] Urken ML, Buchbinder D, Costantino PD, et al. Oromandibular reconstruction using microvascular composite flaps: report of 210 cases. Arch Otolaryngol Head Neck Surg 1998;124:46–55. [5] Hidalgo DA, Pusic AL. Free-flap mandibular reconstruction: a 10-year follow-up study. Plast Reconstr Surg 2002;110:438–49 [discussion 450]. [6] Wax MK, Rosenthal E. Etiology of late free flap failures occurring after hospital discharge. Laryngoscope 2007;117:1961–3. [7] Nuara MJ, Sauder CL, Alam DS. Prospective analysis of outcomes and complications of 300 consecutive microvascular reconstructions. Arch Facial Plast Surg 2009;11:235–9. [8] Suh JD, Sercarz JA, Abemayor E, et al. Analysis of outcome and complications in 400 cases of microvascular head and neck reconstruction. Arch Otolaryngol Head Neck Surg 2004;130:962–6. [9] Deconde AS, Sidell D, Bezouglaia O, Low K, Elashoff D, Grogan T, Tetradis S, Aghaloo T, St. John M. BMP-2 impregnated biomimetic scaffolds successfully induce bone healing in a marginal mandibular defect. Laryngoscope. in press. [10] Levi B, James AW, Nelson ER, et al. Human adipose derived stromal cells heal critical size mouse calvarial defects. PLoS One 2010;5:e11177. [11] Zwetyenga S, Catros A, Emparanza C, et al. Mandibular reconstruction using induced membranes with autologous cancellous bone graft and HA-BTCP: animal model study and preliminary results in patients. Int J Oral Maxillofac Surg 2009;38:1289–97. [12] He Y, Zhang Z, Han-Guang Z, et al. Experimental study on reconstruction of segmental mandible defects using tissue engineered bone combined bone marrow stromal cells with three dimensional tricalcium phosphate. J Craniofac Surg 2007;18:800–5.
[13] Elsalanty ME, Zakhary I, Akeel S, et al. Reconstruction of canine mandibular bone defects using a bone transport reconstruction plate. Ann Plast Surg 2009;63:441–8. [14] Seto I, Asahina I, Oda M, Enomoto S. Reconstruction of the primate mandible with a combination graft of recombinant human bone morphogenic protein-2 and bone marrow. J Oral Maxillofac Surg 2001;59:53–61. [15] Deconde AS, Lee MK, Sidell D, et al. Establishing the critical-sized defect in the rat segmental mandibulectomy animal model. Oral presentation at the AAO-HNSF Annual Meeting, September 2012; 2012. [16] Lee M, Chen TT, Iruela-Arispe ML, Wu BM, Dunn JCY. Modulation of protein delivery from modular polymer scaffolds. Biomaterials 2007;28:1862–70. [17] Johnson KD, Frierson KE, Keller TS, et al. Porous ceramics as bone graft substitutes in long bone defects: a biomechanical, histological, and radiographic analysis. J Orthop Res 1996;14:351–69. [18] Blackwell KE, Azizzadeh B, Ayala C, Rawnsley JD. Octogenarian free flap reconstruction: complications and cost of therapy. Otolaryngol Head Neck Surg 2002;126:301–6. [19] Kang Q, Sun MH, Cheng H, et al. Characterization of the distinct orthotopic bone-forming activity of 14 BMPs using recombinant adenovirus-mediated gene delivery. Gene Ther 2004;11:1312–20. [20] Govender S, Csimma C, Genant HK, Valentin-Opran A, Grp BS. Recombinant human bone morphogenetic protein-2 for treatment of open tibial fractures—a prospective, controlled, randomized study of four hundred and fifty patients. J Bone Joint Surg Am 2002;84A:2123–34. [21] Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 2001;7:211–28. [22] Jeon O, Rhie JW, Kwon I, Kim J, Kim B, Lee S. In vivo bone formation following transplantation of human adipose-derived stromal cells that are not differentiated osteogenically. Tissue Eng A 2008;14:1285–94. [23] Asti A, Gastaldi G, Dorati R, et al. Stem cells grown in osteogenic medium on PLGA, PLGA/HA, and titanium scaffolds for surgical applications. Bioinorg Chem Appl 2010:831031–43. [24] Parrilla C, Saulnier N, Bernardini C, et al. Undifferentiated human adipose tissue-derived stromal cells induce mandibular bone healing in rats. Arch Otolaryngol Head Neck Surg 2011;137:463–70. [25] Schliephake H, Weich HA, Dullin C, Gruber R, Frahse S. Mandibular bone repair by implantation of rhBMP-2 in a slow release carrier of polylactic acid—an experimental study in rats. Biomaterials 2008;29:103–10. [26] Issa JPM, do Nascimento C, Iyomasa MM, et al. Bone healing process in critical-sized defects by rhBMP-2 using poloxamer gel and collagen sponge as carriers. Micron 2008;39:17–24. [27] Arosarena OA, Collins WL. Bone regeneration in the rat mandible with bone morphogenetic protein-2: a comparison of two carriers. Otolaryngol Head Neck Surg 2005;132:592–7. [28] Kruger EA, Im D, Bischoff DS, et al. In vitro mineralization of human mesenchymal stem cells on three-dimensional type I collagen versus PLGA scaffolds: a comparative analysis. Plast Reconstr Surg 2011;127:2301–11.
