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Tyrosine-derived polycarbonate scaffolds for bone regeneration in a rabbit radius critical-size defect model
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2015 Biomed. Mater. 10 035001 (http://iopscience.iop.org/1748-605X/10/3/035001) View the table of contents for this issue, or go to the journal homepage for more
Download details: IP Address: 130.133.8.114 This content was downloaded on 08/05/2015 at 08:32
Please note that terms and conditions apply.
Biomed. Mater. 10 (2015) 035001
doi:10.1088/1748-6041/10/3/035001
Paper
received
2 March 2015
Tyrosine-derived polycarbonate scaffolds for bone regeneration in a rabbit radius critical-size defect model
re vised
5 April 2015 accep ted for publication
7 April 2015 published
8 May 2015
Jinku Kim1,2, Sean McBride1, Amy Donovan1, Aniq Darr3, Maria Hanshella R Magno3 and Jeffrey O Hollinger1 1
Bone Tissue Engineering Center, Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA 15219, USA Department of Bio and Chemical Engineering, Hongik University, Sejong 339–701, Korea 3 Department of Chemistry and Chemical Biology and New Jersey Center for Biomaterials, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA 2
E-mail: [email protected] Keywords: tyrosine-derived polycarbonates, E1001(1k) + CP, bone regeneration, rhBMP-2, rabbit radius critical-size defect, bone tissue engineering
Abstract The aim of the study was to determine bone regeneration in a rabbit radius critical-size defect (CSD) model using a specific polymer composition (E1001(1k)) from a library of tyrosine-derived polycarbonate scaffolds coated with a calcium phosphate (CP) formulation (E1001(1k) + CP) supplemented with recombinant human bone morphogenetic protein-2 (rhBMP-2). Specific doses of rhBMP-2 (0, 17, and 35 μg/scaffold) were used. E1001(1k) + CP scaffolds were implanted in unilateral segmental defects (15 mm length) in the radial diaphyses of New Zealand White rabbits. At 4 and 8 weeks post-implantation, bone regeneration was determined using micro-computed tomography (µCT), histology, and histomorphometry. The quantitative outcome data suggest that E1001(1k) + CP scaffolds with rhBMP-2 were biocompatible and promoted bone regeneration in segmental bone defects. Histological examination of the implant sites showed that scaffolds made of E1001(1k) + CP did not elicit adverse cellular or tissue responses throughout test periods up to 8 weeks. Noteworthy is that the incorporation of a very small amount of rhBMP-2 into the scaffolds (as low as 17 μg/defect site) promoted significant bone regeneration compared to scaffolds consisting of E1001(1k) + CP alone. This finding indicates that E1001(1k) + CP may be an effective platform for bone regeneration in a critical size rabbit radius segmental defect model, requiring only a minimal dose of rhBMP-2.
1. Introduction Although a number of treatment options are currently available for the repair of long bone defects, these therapies have limited effectiveness in large defects and unfavorable side effects remain an issue. Bone autografts, typically harvested from the patient’s iliac crest, are considered the gold standard for the treatment of bone gap defects resulting from trauma, fracture, or tumor resection [1–3]. However, the limited availability of autografts and donor-site morbidity [4] have led to the development of alternative tissue engineering approaches. Combinations of polymeric scaffolds treated with biomolecules such as recombinant human bone morphogenetic protein-2 (rhBMP-2) or rhBMP-7 and/or stem cells (e.g. mesenchymal stem cells, MSCs) have gained popularity [5]. Consequently, tissue engineering-based products such as absorbable © 2015 IOP Publishing Ltd
collagen sponges (ACS) in combination with rhBMP-7 or recombinant human osteogenic protein-1 (rhOP-1, Stryker Biotech, Kalamazoo, MI) have been approved by the US FDA [6, 7] However, clinical complications with ACS (e.g. rapid protein degradation or soft tissue inflammation) have been reported [8] and ACS lacks versatility to control pharmacokinetics of expensive biomolecules. In addition, frequently used delivery systems such as poly(α-hydroxy acid)-based scaffolds may produce unfavorable degradation products leading to inflammatory responses [2, 9]. Furthermore, increasing doses of rhBMP-2 often trigger lipid formation as a result of adipogenic stimulation, or ectopic bone growth, which are serious risk factors. Putative etiology for adverse clinical outcomes may be due to supraphysiological rhBMP doses and carrier insufficiency. Thus, patient safety issues have prompted concern from the FDA and from clinicians [10, 11].
Biomed. Mater. 10 (2015) 035001
J Kim et al
Tyrosine-derived polycarbonates have been developed as biomaterials by substituting cytotoxic bisphenol-A monomer with natural L-tyrosine amino acid-derived monomers, which have excellent biocompatibility, while maintaining strong mechanical properties of engineering plastics (i.e. polycarbonates) [12, 13]. Recently, the laboratory of Joachim Kohn at Rutgers University has developed a new class of tyrosine-derived polycarbonates (TyrPCs), which is composed of desaminotyrosyl-tyrosine alkyl ester (DTR), desaminotyrosyl-tyrosine (DT), and poly(ethylene glycol) (PEG) [14]. By incorporating those molecules into the polymer backbone, the properties of the resulting tyrosine-derived terpolymers were fine-tuned [14] Among numerous compositions of TyrPCs, E1001(1k) has emerged as an useful platform for bone tissue engineering scaffold regarding and in vitro and in vivo biological outcomes [15, 16]. E1001(1k) is a notation for poly(DTE-co-10%-DT-co-1%-PEG1k carbonate) where DTE is desaminotyrosyl-tyrosine ethyl ester, 10% is the mole percent of DT, 1% is the mole percent of PEG, and 1k (or 1,000) is the weight average molecular weight of PEG [14]. In those studies, different scaffold formulations of E1001(1k) with or without additional calcium phosphate (CP) minerals and/ or rhBMP-2 were explored in the rabbit calvaria model [16]. To date, the impact of rhBMP-2 in combination with TyrPC-based scaffolds on in vivo performance in a more challenging rabbit radius critical size defect (CSD) model has not been investigated. This report is part of a series of studies using E1001(1k) as a scaffold material [15, 16]. Since a previous study showed better bone regeneration potential of E1001(1k) coated with calcium phosphate [16], we report here on the use of E1001(1k) + CP scaffolds in the rabbit radius model and seek to determine an optimal rhBMP-2 dose that maximally promotes bone regeneration without undesirable side effects in a rabbit segmental defect. The purpose of this study was to extend our studies of E1001(1k) + CP scaffolds to the rabbit radius model. As part of this study, we hypothesized that a E1001(1k) + CP scaffold in the rabbit radius model will promote bone regeneration in the defect without unfavorable tissue responses. We also determined the effects of scaffold supplementation with 0, 17, or 35 μg rhBMP-2 on bone regeneration in the rabbit radius model. In this study, E1001(1k) + CP scaffolds were surgically implanted in the CSD rabbit radius model and the outcomes were determined via mirco computed tomography (µCT), histological, and histomorphometric analyses.
2. Materials and methods 2.1. Scaffold preparation Cylinder-shaped porous E1001(1k) scaffolds (5 mm diameter × 15 mm length) were produced using a combination of solvent casting, porogen leaching, and phase separation techniques as described previously for disk-shaped scaffolds [14]. Briefly, 300 mg of the 2
Table 1. Experimental design (number of rabbits per treatment group). Treatment
Time period (weeks)
Total
4
8
0 µg rhBMP-2
5
5
10
17 µg
5
5
10
35 µg
5
5
10
Total
15
15
30
polymer (molecular weight: 271 kDa) were dissolved in 300 μL of DI water and 3 mL of 1,4-dioxane and then the polymer solution was poured into a Teflon dish containing NaCl porogen (200–400 μm particle size). After 1 h of diffusion, the mold was frozen in liquid nitrogen and then freeze-dried for 48 h. Dried scaffolds were cut into cylindrical shape using a custom-made stainless steel puncher and incubated in DI water until porogens were fully leached out. The scaffolds were further dried for 48 h under vacuum at room temperature. E1001(1k) + CP scaffolds were prepared by incubating the scaffolds in 1 M CaCl2 solution followed by 0.96 M K2HPO4 solution with vortexing at 200 rpm. This resulted in the formation of a dicalcium phosphate dihydrate (DCPD, CaHPO 4 ·2H 2 O) precipitates within the pores of the scaffold. Purity of the composition of calcium phosphate coatings on the scaffold were confirmed by Fourier Transform infrared spectroscopy and x-ray diffraction analyses. Pore structure of the scaffolds were determined using a scanning electron microscope (SEM, Amray 1830I, 20 kV). A known doses of rhBMP-2 (0, 17, and 35 μg) (Wyeth, Cambridge, MA) in 170 μL of buffer were added dropwise to each scaffold (100 mg). The selected volume (170 μL) was the maximum fluid volume, which could be maintained in the void volume of the E1001(1k) + CP scaffolds. The scaffolds were air-dried overnight under sterile conditions and aseptically packaged for surgery. 2.2. Rabbit radius surgery Critical-sized ostectomy gaps (15 mm in length) were prepared in radii of skeletally mature New Zealand White rabbits (Myrtles Rabbitary Inc., Thompson Station, TN) aged between 6 and 18 months and weighing 3.5–4.5 kg (n = 5 rabbits/experimental group) (table 1). Surgical procedures were approved by the institutional animal care and use committee (IACUC) at the Allegheny-Singer Research Institute in Pittsburgh, PA as well as animal care and use review office (ACURO) at the Department of Army. The NIH Guide for Care and Use of Laboratory Animals was observed in the treatment of all experimental animals. Surgical research was conducted in compliance with the Animal Welfare Act and other Federal statutes relating to animals and experiments involving animals and as per principles set forth in the Guide for the Care and Use of Laboratory Animals, National Research Council (1996).
Biomed. Mater. 10 (2015) 035001
J Kim et al
Aseptic surgical technique previously described was used to prepare the ostectomy gap [2]. A critical-sized defect 15 mm in length in the diaphysis of the radius (randomly selected, unilateral, right or left) was prepared in the rabbits using a surgical saw and copious physiologic saline. Anesthesia was induced with an intramuscular injection within 30 min of premedication using glycopyrrolate at 0.01 mg kg−1 and ketamine at 40–80 mg kg−1 IM. Following induction, an auricular IV catheter was put in place. Ophthalmic ointment will be gently placed on the corneas. Rabbits were intubated (3–3.5 ETT) and placed on 2–3% isoflurane anesthesia delivered in 100% oxygen using a non-rebreathing circuit. Each rabbit received Lactated Ringer’s solution at 10 ml kg−1 h−1 throughout anesthesia and enroflaxacin antibiotics (Baytril at 5 mg kg−1, SQ), 1 h prior to surgery. The surgical site (right or left front limb) was clipped using an electric clipper and prepared for aseptic surgery (3-chlorhexidine [4%] scrubs alternating with sterile saline solution). The paw of the operative limb was enclosed in a sterile surgical glove and the rabbit draped. A tourniquet was placed gently at the axilla and a 2.5 cm incision was made over the middle of the radius and the bone was exposed by sharp dissection through overlying skin. Each surgery was performed using standard sterile orthopedic packs and equipment. A 15 mm in length section of the diaphysis was removed with a surgical saw using copious sterile physiologic saline irrigation. Following hemostasis, the test articles (scaffolds) were inserted into the defect. The synchondrosis between the radius and ulna proximal and distal to the ostectomized diaphysis provides adequate fixation. Soft tissue closure was accomplished using 4.0 Dexon suture in a simple interrupted fashion and thereafter the tourniquet was taken down. At 4 and 8 weeks post-implantation, all animals were euthanized humanely according to the approved IACUC protocol using an overdose of barbiturate solution (Euthanasia solution; 2.2 mg 10 kg −1 IV). At necropsy, the sites of surgical implantation with a surrounding margin of host tissues were harvested and placed immediately in neutralized formalin buffer. The implant and surrounding tissues were harvested and prepared for micro-CT, histology, and histomorphometry. 2.3. Micro-computed tomography (µCT), histology and histomorphometry The new bone formation in harvested radius specimens were imaged by µCT and bone volume and bone coverage were quantified in previously described manner [17, 18]. Briefly, Each specimen was analyzed by a µCT imaging system (GE Healthcare, Piscataway, NJ) with 20 µm resolution (80 kVp, 500 mA, 26 min total scan time). The 3D data were processed and rendered (isosurface/maximum intensity projections) using MicroView (GE Healthcare). Trabecular bone volume in a defect site was calculated using the image 3
analysis (MicroView, GE Healthcare) of µCT data. Briefly, after 3D reconstruction, each volume was scaled to hounsfield units (HU) using a calibration phantom containing air and water. Since each volume was calibrated using a fixed standard, calcium phosphate, cortical bone, trabecular/woven bone, and scaffold content was determined using predefined Hounsfield Unit thresholds (>3000, 2000–3000, 750–2000, and 300–750 respectively). Bone coverage was calculated by a pseudoradiograph obtained by a projected image of the radius (in superior–inferior direction) from the 3D µCT data set [18]. For histology analysis, implant sites were prepared for undecalcified plastic embedding and were stained with Sanderson’s Rapid Bone Stain and counterstained with van Gieson’s picrofuchsin [14]. Briefly, the recipient bone and implants (referred to collectively as specimens) were dehydrated in ascending grades of ethanol, cleared in xylene at 4 °C to minimize implant solvation during the processing and embedded in poly(methyl methacrylate). The specimens were cut and ground to 30 µm thick sections with an Exakt diamond band saw and MicroGrinder (Exakt Technologies, Oklahoma City, OK). The slides were prepared and stained with Sanderson’s Rapid Bone Stain and counterstained with van Gieson’s picrofuchsin, which resulted in soft tissue staining blue and bone staining pink/red. New bone formation within the defect was measured by histomorphometry using an image analysis program (ImagePro version 7.0, Media Cybernetics, Bethesda, MD). Briefly, the defect area (region of interest, ROI) on each histology section (1.5×) was selected and the areas of new bone were determined based on predetermined color thresholds. The percentage of new bone area was obtained by dividing the bone area by whole defect area. 2.4. Statistics All data are reported as a mean ± standard deviation and tested for significance (p
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My IOPscience
Tyrosine-derived polycarbonate scaffolds for bone regeneration in a rabbit radius critical-size defect model
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2015 Biomed. Mater. 10 035001 (http://iopscience.iop.org/1748-605X/10/3/035001) View the table of contents for this issue, or go to the journal homepage for more
Download details: IP Address: 130.133.8.114 This content was downloaded on 08/05/2015 at 08:32
Please note that terms and conditions apply.
Biomed. Mater. 10 (2015) 035001
doi:10.1088/1748-6041/10/3/035001
Paper
received
2 March 2015
Tyrosine-derived polycarbonate scaffolds for bone regeneration in a rabbit radius critical-size defect model
re vised
5 April 2015 accep ted for publication
7 April 2015 published
8 May 2015
Jinku Kim1,2, Sean McBride1, Amy Donovan1, Aniq Darr3, Maria Hanshella R Magno3 and Jeffrey O Hollinger1 1
Bone Tissue Engineering Center, Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA 15219, USA Department of Bio and Chemical Engineering, Hongik University, Sejong 339–701, Korea 3 Department of Chemistry and Chemical Biology and New Jersey Center for Biomaterials, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA 2
E-mail: [email protected] Keywords: tyrosine-derived polycarbonates, E1001(1k) + CP, bone regeneration, rhBMP-2, rabbit radius critical-size defect, bone tissue engineering
Abstract The aim of the study was to determine bone regeneration in a rabbit radius critical-size defect (CSD) model using a specific polymer composition (E1001(1k)) from a library of tyrosine-derived polycarbonate scaffolds coated with a calcium phosphate (CP) formulation (E1001(1k) + CP) supplemented with recombinant human bone morphogenetic protein-2 (rhBMP-2). Specific doses of rhBMP-2 (0, 17, and 35 μg/scaffold) were used. E1001(1k) + CP scaffolds were implanted in unilateral segmental defects (15 mm length) in the radial diaphyses of New Zealand White rabbits. At 4 and 8 weeks post-implantation, bone regeneration was determined using micro-computed tomography (µCT), histology, and histomorphometry. The quantitative outcome data suggest that E1001(1k) + CP scaffolds with rhBMP-2 were biocompatible and promoted bone regeneration in segmental bone defects. Histological examination of the implant sites showed that scaffolds made of E1001(1k) + CP did not elicit adverse cellular or tissue responses throughout test periods up to 8 weeks. Noteworthy is that the incorporation of a very small amount of rhBMP-2 into the scaffolds (as low as 17 μg/defect site) promoted significant bone regeneration compared to scaffolds consisting of E1001(1k) + CP alone. This finding indicates that E1001(1k) + CP may be an effective platform for bone regeneration in a critical size rabbit radius segmental defect model, requiring only a minimal dose of rhBMP-2.
1. Introduction Although a number of treatment options are currently available for the repair of long bone defects, these therapies have limited effectiveness in large defects and unfavorable side effects remain an issue. Bone autografts, typically harvested from the patient’s iliac crest, are considered the gold standard for the treatment of bone gap defects resulting from trauma, fracture, or tumor resection [1–3]. However, the limited availability of autografts and donor-site morbidity [4] have led to the development of alternative tissue engineering approaches. Combinations of polymeric scaffolds treated with biomolecules such as recombinant human bone morphogenetic protein-2 (rhBMP-2) or rhBMP-7 and/or stem cells (e.g. mesenchymal stem cells, MSCs) have gained popularity [5]. Consequently, tissue engineering-based products such as absorbable © 2015 IOP Publishing Ltd
collagen sponges (ACS) in combination with rhBMP-7 or recombinant human osteogenic protein-1 (rhOP-1, Stryker Biotech, Kalamazoo, MI) have been approved by the US FDA [6, 7] However, clinical complications with ACS (e.g. rapid protein degradation or soft tissue inflammation) have been reported [8] and ACS lacks versatility to control pharmacokinetics of expensive biomolecules. In addition, frequently used delivery systems such as poly(α-hydroxy acid)-based scaffolds may produce unfavorable degradation products leading to inflammatory responses [2, 9]. Furthermore, increasing doses of rhBMP-2 often trigger lipid formation as a result of adipogenic stimulation, or ectopic bone growth, which are serious risk factors. Putative etiology for adverse clinical outcomes may be due to supraphysiological rhBMP doses and carrier insufficiency. Thus, patient safety issues have prompted concern from the FDA and from clinicians [10, 11].
Biomed. Mater. 10 (2015) 035001
J Kim et al
Tyrosine-derived polycarbonates have been developed as biomaterials by substituting cytotoxic bisphenol-A monomer with natural L-tyrosine amino acid-derived monomers, which have excellent biocompatibility, while maintaining strong mechanical properties of engineering plastics (i.e. polycarbonates) [12, 13]. Recently, the laboratory of Joachim Kohn at Rutgers University has developed a new class of tyrosine-derived polycarbonates (TyrPCs), which is composed of desaminotyrosyl-tyrosine alkyl ester (DTR), desaminotyrosyl-tyrosine (DT), and poly(ethylene glycol) (PEG) [14]. By incorporating those molecules into the polymer backbone, the properties of the resulting tyrosine-derived terpolymers were fine-tuned [14] Among numerous compositions of TyrPCs, E1001(1k) has emerged as an useful platform for bone tissue engineering scaffold regarding and in vitro and in vivo biological outcomes [15, 16]. E1001(1k) is a notation for poly(DTE-co-10%-DT-co-1%-PEG1k carbonate) where DTE is desaminotyrosyl-tyrosine ethyl ester, 10% is the mole percent of DT, 1% is the mole percent of PEG, and 1k (or 1,000) is the weight average molecular weight of PEG [14]. In those studies, different scaffold formulations of E1001(1k) with or without additional calcium phosphate (CP) minerals and/ or rhBMP-2 were explored in the rabbit calvaria model [16]. To date, the impact of rhBMP-2 in combination with TyrPC-based scaffolds on in vivo performance in a more challenging rabbit radius critical size defect (CSD) model has not been investigated. This report is part of a series of studies using E1001(1k) as a scaffold material [15, 16]. Since a previous study showed better bone regeneration potential of E1001(1k) coated with calcium phosphate [16], we report here on the use of E1001(1k) + CP scaffolds in the rabbit radius model and seek to determine an optimal rhBMP-2 dose that maximally promotes bone regeneration without undesirable side effects in a rabbit segmental defect. The purpose of this study was to extend our studies of E1001(1k) + CP scaffolds to the rabbit radius model. As part of this study, we hypothesized that a E1001(1k) + CP scaffold in the rabbit radius model will promote bone regeneration in the defect without unfavorable tissue responses. We also determined the effects of scaffold supplementation with 0, 17, or 35 μg rhBMP-2 on bone regeneration in the rabbit radius model. In this study, E1001(1k) + CP scaffolds were surgically implanted in the CSD rabbit radius model and the outcomes were determined via mirco computed tomography (µCT), histological, and histomorphometric analyses.
2. Materials and methods 2.1. Scaffold preparation Cylinder-shaped porous E1001(1k) scaffolds (5 mm diameter × 15 mm length) were produced using a combination of solvent casting, porogen leaching, and phase separation techniques as described previously for disk-shaped scaffolds [14]. Briefly, 300 mg of the 2
Table 1. Experimental design (number of rabbits per treatment group). Treatment
Time period (weeks)
Total
4
8
0 µg rhBMP-2
5
5
10
17 µg
5
5
10
35 µg
5
5
10
Total
15
15
30
polymer (molecular weight: 271 kDa) were dissolved in 300 μL of DI water and 3 mL of 1,4-dioxane and then the polymer solution was poured into a Teflon dish containing NaCl porogen (200–400 μm particle size). After 1 h of diffusion, the mold was frozen in liquid nitrogen and then freeze-dried for 48 h. Dried scaffolds were cut into cylindrical shape using a custom-made stainless steel puncher and incubated in DI water until porogens were fully leached out. The scaffolds were further dried for 48 h under vacuum at room temperature. E1001(1k) + CP scaffolds were prepared by incubating the scaffolds in 1 M CaCl2 solution followed by 0.96 M K2HPO4 solution with vortexing at 200 rpm. This resulted in the formation of a dicalcium phosphate dihydrate (DCPD, CaHPO 4 ·2H 2 O) precipitates within the pores of the scaffold. Purity of the composition of calcium phosphate coatings on the scaffold were confirmed by Fourier Transform infrared spectroscopy and x-ray diffraction analyses. Pore structure of the scaffolds were determined using a scanning electron microscope (SEM, Amray 1830I, 20 kV). A known doses of rhBMP-2 (0, 17, and 35 μg) (Wyeth, Cambridge, MA) in 170 μL of buffer were added dropwise to each scaffold (100 mg). The selected volume (170 μL) was the maximum fluid volume, which could be maintained in the void volume of the E1001(1k) + CP scaffolds. The scaffolds were air-dried overnight under sterile conditions and aseptically packaged for surgery. 2.2. Rabbit radius surgery Critical-sized ostectomy gaps (15 mm in length) were prepared in radii of skeletally mature New Zealand White rabbits (Myrtles Rabbitary Inc., Thompson Station, TN) aged between 6 and 18 months and weighing 3.5–4.5 kg (n = 5 rabbits/experimental group) (table 1). Surgical procedures were approved by the institutional animal care and use committee (IACUC) at the Allegheny-Singer Research Institute in Pittsburgh, PA as well as animal care and use review office (ACURO) at the Department of Army. The NIH Guide for Care and Use of Laboratory Animals was observed in the treatment of all experimental animals. Surgical research was conducted in compliance with the Animal Welfare Act and other Federal statutes relating to animals and experiments involving animals and as per principles set forth in the Guide for the Care and Use of Laboratory Animals, National Research Council (1996).
Biomed. Mater. 10 (2015) 035001
J Kim et al
Aseptic surgical technique previously described was used to prepare the ostectomy gap [2]. A critical-sized defect 15 mm in length in the diaphysis of the radius (randomly selected, unilateral, right or left) was prepared in the rabbits using a surgical saw and copious physiologic saline. Anesthesia was induced with an intramuscular injection within 30 min of premedication using glycopyrrolate at 0.01 mg kg−1 and ketamine at 40–80 mg kg−1 IM. Following induction, an auricular IV catheter was put in place. Ophthalmic ointment will be gently placed on the corneas. Rabbits were intubated (3–3.5 ETT) and placed on 2–3% isoflurane anesthesia delivered in 100% oxygen using a non-rebreathing circuit. Each rabbit received Lactated Ringer’s solution at 10 ml kg−1 h−1 throughout anesthesia and enroflaxacin antibiotics (Baytril at 5 mg kg−1, SQ), 1 h prior to surgery. The surgical site (right or left front limb) was clipped using an electric clipper and prepared for aseptic surgery (3-chlorhexidine [4%] scrubs alternating with sterile saline solution). The paw of the operative limb was enclosed in a sterile surgical glove and the rabbit draped. A tourniquet was placed gently at the axilla and a 2.5 cm incision was made over the middle of the radius and the bone was exposed by sharp dissection through overlying skin. Each surgery was performed using standard sterile orthopedic packs and equipment. A 15 mm in length section of the diaphysis was removed with a surgical saw using copious sterile physiologic saline irrigation. Following hemostasis, the test articles (scaffolds) were inserted into the defect. The synchondrosis between the radius and ulna proximal and distal to the ostectomized diaphysis provides adequate fixation. Soft tissue closure was accomplished using 4.0 Dexon suture in a simple interrupted fashion and thereafter the tourniquet was taken down. At 4 and 8 weeks post-implantation, all animals were euthanized humanely according to the approved IACUC protocol using an overdose of barbiturate solution (Euthanasia solution; 2.2 mg 10 kg −1 IV). At necropsy, the sites of surgical implantation with a surrounding margin of host tissues were harvested and placed immediately in neutralized formalin buffer. The implant and surrounding tissues were harvested and prepared for micro-CT, histology, and histomorphometry. 2.3. Micro-computed tomography (µCT), histology and histomorphometry The new bone formation in harvested radius specimens were imaged by µCT and bone volume and bone coverage were quantified in previously described manner [17, 18]. Briefly, Each specimen was analyzed by a µCT imaging system (GE Healthcare, Piscataway, NJ) with 20 µm resolution (80 kVp, 500 mA, 26 min total scan time). The 3D data were processed and rendered (isosurface/maximum intensity projections) using MicroView (GE Healthcare). Trabecular bone volume in a defect site was calculated using the image 3
analysis (MicroView, GE Healthcare) of µCT data. Briefly, after 3D reconstruction, each volume was scaled to hounsfield units (HU) using a calibration phantom containing air and water. Since each volume was calibrated using a fixed standard, calcium phosphate, cortical bone, trabecular/woven bone, and scaffold content was determined using predefined Hounsfield Unit thresholds (>3000, 2000–3000, 750–2000, and 300–750 respectively). Bone coverage was calculated by a pseudoradiograph obtained by a projected image of the radius (in superior–inferior direction) from the 3D µCT data set [18]. For histology analysis, implant sites were prepared for undecalcified plastic embedding and were stained with Sanderson’s Rapid Bone Stain and counterstained with van Gieson’s picrofuchsin [14]. Briefly, the recipient bone and implants (referred to collectively as specimens) were dehydrated in ascending grades of ethanol, cleared in xylene at 4 °C to minimize implant solvation during the processing and embedded in poly(methyl methacrylate). The specimens were cut and ground to 30 µm thick sections with an Exakt diamond band saw and MicroGrinder (Exakt Technologies, Oklahoma City, OK). The slides were prepared and stained with Sanderson’s Rapid Bone Stain and counterstained with van Gieson’s picrofuchsin, which resulted in soft tissue staining blue and bone staining pink/red. New bone formation within the defect was measured by histomorphometry using an image analysis program (ImagePro version 7.0, Media Cybernetics, Bethesda, MD). Briefly, the defect area (region of interest, ROI) on each histology section (1.5×) was selected and the areas of new bone were determined based on predetermined color thresholds. The percentage of new bone area was obtained by dividing the bone area by whole defect area. 2.4. Statistics All data are reported as a mean ± standard deviation and tested for significance (p
