Abstract
Objective
To investigate the efficacy of biomimetic PLGA scaffolds, alone and in 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 semi-quantitative 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 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.
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 drug-induced osteonecrosis, or as a complication of traumatic injury . 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 . Currently, microvascular free tissue transfer is considered the gold standard for oromandibular reconstruction . However, free flap surgery is lengthy and is associated with perioperative complication rates affecting up to 30%–40% of patients .
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(lactic-co-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 adipose-derived stem cells (multipotent stem cells with the potential for ostegenic differentiation) . Animal studies have been performed utilizing a variety of animal models, including canine, primate, and rabbit . 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 and established 5-mm as the critical-sized defect, i.e. defect size that will not heal spontaneously .
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 environment (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; Sigma-Aldrich, 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 . In brief, PLGA/chloroform solutions were mixed with 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 H 2 O, 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 H 2 O. 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 × 10 7 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 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 . 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 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).
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 environment (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; Sigma-Aldrich, 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 . In brief, PLGA/chloroform solutions were mixed with 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 H 2 O, 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 H 2 O. 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 × 10 7 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.
