Biomaterials 35 (2014) 236e248
Contents lists available at ScienceDirect
Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Injectable thermosensitive PEGePCLePEG hydrogel/acellular bone matrix composite for bone regeneration in cranial defects PeiYan Ni, QiuXia Ding, Min Fan, JinFeng Liao, ZhiYong Qian*, JingCong Luo*, XiuQun Li, Feng Luo, ZhiMing Yang, YuQuan Wei State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, Chengdu, 610041, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 September 2013 Accepted 2 October 2013 Available online 17 October 2013
We have certified that the injectable thermosensitive ABM/PECE composite presented promising potential in bone regeneration benefited from the incorporation of the intrinsic osteoinductive acellular bone matrix (ABM) granules into the poly(ethylene glycol)epoly(ε-capro-lactone)epoly(ethylene glycol) (PEGePCLePEG, PECE) hydrogel. In this study, the 12 mm 8 mm 2 mm cranial defects of the New Zealand white rabbits were fabricated to evaluate the bone regeneration effect. The ABM/PECE composite was injected into the defect while the pure PECE as control, and the bone regeneration was evaluated at 4, 12 and 20 weeks post-surgery by X-radiological examination, micro-computed tomography examination and histological analysis. In ABM/PECE composite treated group, the new bone formed originally from both the margin and the center of the defect, and the defect region had healed up to 20 weeks. Furthermore, the shadow density of the newly formed bone eventually approximated to host cortical bone. In comparison, the control group was filled with sparing new bone with low-density only from the periphery of the defect. Meanwhile, the quantitative determination of new bone by histomorphometry confirmed the excellent bone regeneration of ABM/PECE composite. All the results suggested that the ABM/PECE composite presented enhanced bone regeneration guidance in rabbit cranial defects. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Acellular bone matrix/PEGePCLePEG (ABM/ PECE) Bone regeneration Injectable Thermosensitive hydrogel Cranial defects
1. Introduction The cranial defects resulted from abnormalities, trauma, infection or tumor resection require closure for both functional and esthetic reasons, which have posed great challenge to bone repair materials research and clinical surgeries in the world [1e4]. It is favored to pay close attention to designing and engineering materials of similar composition, structure and properties with the native bone extracellular matrix (ECM) to reconstruct the degenerative or defective cranial bones. During the past several decades, a range of biomaterials have been investigated as bone substitutions. According to the composition, the bone substitutions can be classified mainly into three categories: biological source bone graft, synthesized degradable or undegradable materials and composites of them. The biological source bone graft have been proposed based on their similar composition and structure with the natural ECM, excellent bioactivity, non-cytotoxicity and appropriate strength, * Corresponding authors. Tel.: þ86 28 85164063; fax: þ86 28 85164060. E-mail addresses: [email protected], [email protected] (Z. Qian), [email protected] (J. Luo). 0142-9612/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2013.10.016
including autologous bones [5], allografts [6] and xenografts [7]. However, the application of autologous bones and allografts has been limited in the amount of bone available, potential problems associated with the secondary wound, and difficulties in prefabricating suitable shape and depth to mold the cranial defects cavity [1]. During the past decades, acellular bone matrix (ABM) has attracted increasingly attention as a substitute for recipient bone tissue [8e10]. Besides the advantages described above, ABM with low-immunogenicity has greatly widened source benefited from the acellular operation during the preparation. Therefore, the optimized ABM have been successfully applied in multiple animal models [11e13], and the processed ABM informed the potential to support osteoinduction [14,15]. Furthermore, the ABM can be grounded into granules and prefabricated almost right to mold the cavity even though the bone defects come in a variety of shapes and depths [16,17]. However, the brittle ABM granules might migrate out of the defect cavity and induce severe inflammation, which might bring great pain to the patients [18e20]. One promising strategy to overcome the brittleness of ABM granules is to introduce a optimized polymeric component as a 3D scaffold [21]. Up to now, several biodegradable polymers have been proven to be acceptable in the bone regeneration field to
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some degree [22,23]. Among them, the injectable hydrogel polymers have been increasingly applicated because of easy administration, minimally invasive procedures, site specific introduction, patient convenience and three-dimensional (3D) network formation under mild conditions [24e27]. In particular, thermosensitive hydrogel could be injected freely at room temperature or lower, and form a gel depot in situ to take the shape of the cavity in the impaired tissue especially at the body temperature. Besides, the thermosensitive hydrogels give a promising modality to avoid using any noxious substances as initiator of phase transition (such as the pH adjustments, oxidants or UV light) owing to the sensitivity to the temperature. Among the intelligent thermosensitive hydrogel, poly(ethylene glycol)poly(ε-capro-lactone)-poly(ethylene glycol) (PEGePCLePEG, PECE) is a linear triblock polyester copolymer composed of hydrophilic poly(ethylene glycol) (PEG) block and hydrophobic poly(ε-capro-lactone) (PCL) block [28e30]. As well known, both PEG and PCL are FDA-approved biomaterials and they have been widely used in biomedical field [31e33]. The physicalechemical properties [34], drug delivery behavior [35], the biodegradability as well as non-cytotoxicity both in vitro and in vivo [30,35] of PECE copolymer have been comprehensively studied in our previous works. In previous works of our group [29], the ABM/PECE hydrogel composite was fabricated by incorporating the bioactive ABM into the biodegradable thermosensitive hydrogel PECE, which could overcome the brittleness of ABM granules. As indicated, ABM/PECE hydrogel composite showed no significant cytotoxicity to rat bone mesenchymal stem cells (MSCs) in vitro and were non-cytotoxic in the BALB/c mice subcutis even up to 4 weeks. Based on the above mentioned investigations, we prospect that the ABM/PECE hydrogel composite could facilitate the bone regeneration in the non-load-bearing cranial repair process by combining the advantages of the intrinsic osteoinductive ABM granules and the injectable thermosensitive PECE hydrogel. The aim of this study was to extend the in vitro results to the in vivo setting by evaluating the effects of ABM/PECE composite in repairing the cranial defects of New Zealand white rabbits. X-ray examination, 3D microcomputed tomography images analysis and histological analysis were performed to evaluate the newly formed bone. 2. Materials and methods 2.1. Materials The (ABM) was prepared with physical and chemical methods as depicted before [36,37]. A section of fresh human femur without soft tissue was soaked in 3% H2O2 to oxidize proteins and reduce antigenicity. After treatment bath, the sectioned bone scaffolds were immersed in the defatting solvent to remove remaining marrow and intramatrix cells. The as-obtained ABM were freeze-dried and ground to granules, the shape and size of which were irregular (1e20 mm). Then the prepared ABM granules were stored in sterile bags at room temperature. Poly(ethylene glycol)poly(ε-capro-lactone)-poly(ethylene glycol) (PEGePCLePEG, PECE) copolymers used in this study were synthesized in two steps as described in previous works briefly [28,29,31]. The number-average molecular weight (Mn) of as-obtained PECE copolymer was 3150, which was determined by the integral intensities of characteristic absorption peaks at 3.60 and 4.07 ppm in the 1H NMR spectrum [38]. After incorporating ABM granules into the PECE hydrogel, the obtained ABM/PECE hydrogel composite (30 wt % ABM) was sterilized by 60Co gamma rays using a set dose of 30 kGy. Other chemical agents like CH2Cl2, petroleum ether, and chloroform were obtained from Chengdu Kelong Chemicals, China. All of them were analytical reagent and used directly without further purification. 2.2. Characterizations of ABM/PECE composite 2.2.1. X-ray diffraction (XRD) X-ray diffraction measurements were used to study the phase analysis and crystallinity of the ABM/PECE composite samples using an X’Pert Pro MPD DY1291 (PHILIPS, Netherlands) diffractometer. The X-ray diffraction was studied using graphite monochromatized Cu K radiation (l ¼ 0.1542 nm; 35 mA; 40 kV) in the 2q ranging from 10 to 60 at a scanning rate of 4 /min.
Fig. 1. X-ray diffraction patterns of pure PECE copolymer (A), ABM granules (B) and ABM/PECE composite (C).
2.2.2. Differential scanning calorimetry (DSC) A differential scanning calorimeter (DSC; NETSCZ 204, NETSCZ, Germany) was used to study the thermal properties of the ABM/PECE hydrogel composite. The dry samples (about 5.0 mg) were loaded in a cell and heat exchange was recorded during the heating and cooling cycle. The samples were firstly heated from room temperature to 100 C under nitrogen atmosphere then cooled to 20 C at a heating rate of 10 C/min, and reheated to 100 C again at the same rate.
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2.2.3. Solegelesol phase transition behavior study Solegelesol phase transition behavior of ABM/PECE hydrogel composite was investigated using test tube-inverting method [34,35]. The sol was defined as ‘flow transparent liquid’ while the gel was defined as ‘no flow opaque solid’, respectively. The solegelesol transition was visually observed by inverting the vials, and the state of the composite held in 1 min. In this study, the hydrogel composite were firstly transited from 10 C to physiological temperature of 37 C, and then back to 10 C. 2.3. Toxicity evaluation in vitro and in vivo 2.3.1. Cytotoxicity, cell proliferation and cell cycle evaluation of ABM/PECE hydrogel composite by WST-1 and flow cytometry The leachates were extracted from ABM/PECE hydrogel composite (30 wt % ABM) for 24 h using DMEM. The sequential dilutions of the stock leachates (100%) were carried out to vary the concentrations (100%, 50%, 25%) [29]. The rat ROS 17/2.8 osteoblasts were incubated with the leachates of different concentrations (0%, 25%, 50%, 100%, n ¼ 3) up to 5 d. The effects of ABM/PECE hydrogel on cytotoxicity and cell proliferation were evaluated by WST-1 method and the absorbance at 570 nm was measured at predetermined 1d, 3d and 5d. Meanwhile, the treated cells were washed twice with PBS and fixed with 70% ethanol after harvest at day 3. The fixed cells were stained with PI solution (50 mg/ml) following RNase A (1 mg/ml) treated, and the cell cycles were analyzed by flow cytometry according to the content of DNA. 2.3.2. Implantation of ABM/PECE hydrogel composite into muscle pouches of rats All the animal experiment protocols were approved by the Institutional Animal Care and Use Committee and were in compliance with all regulatory guidelines. An incision was made at the dorsal sites of the hind legs after the rat was anesthetized with pentobarbital sodium (30 mg/kg, i.p.) and sterilized with iodophor and 70% ethanol solution. The muscle pouches in the dorsal sites were exposed with a tweezer and 0.5 ml of ABM/PECE hydrogel composite was injected. After the operation, the muscle pouch and incision of skin were sewed up respectively and all rats received intramuscular injection of 400,000 units’ penicillin in the following 3 days. The rats were sacrificed at week 2 and week 4 post-surgery, and the implants together with surrounding tissues were harvested and fixed immediately with 10% neutral formalin. Then the samples were embedded in paraffin after a graded dehydration. The sections with a thickness of 5 mm were made by LEICA 2500E diamond saw microtome (Leica SpA, Milan, Italy) and stained according to standard protocols for hematoxylin & eosin (H&E). The sections were analyzed with a digital image analysis system (Nikon E 600 Microscope with a Nikon Digital Camera DXM 1200, Nikon Corporation, Japan). 2.4. Cranial defects construction and bone regeneration evaluation 2.4.1. Surgical procedure To evaluate the bone regeneration, the ABM/PECE hydrogel composite was transplanted into the cranial defects of the New Zealand white rabbits. New Zealand white rabbits (3.0 0.2 kg) used in this study were purchased from the Experimental Animals Center of Sichuan Province, China. All the protocols were approved by the Institutional Animal Care and Use Committee of West China Hospital. Briefly, each New Zealand white rabbits were anaesthetized with pentobarbital sodium (30 mg/kg, i.p.) until the effective anesthesia. The surgical area was shaved and prepared with povidone-iod for aseptic surgery. The calvaria bone was exposed after a straight incision approximately 5 cm in length from the nasal bone to the midsaggital crest, and the periosteum was also incised in the same place. Two rectangular 12 mm 8 mm 2 mm defects were made by a ground section equipped-in a
dental hand piece with copious physiologic saline irrigation as coolant to remove bone debris. The defects were made on each side of the median sagittal suture and care was exercised to avoid injury of the dura [39]. Subsequently, the right calvaria defect was filled with prepared ABM/PECE composite solution while the left defect was filled with PECE hydrogel as control. After placement of the materials, the soft tissues were closed with sutures. All the rabbits received intramuscular injection of 400,000 units’ penicillin per day during the first week post-operation. Meanwhile, their general conditions were observed continuously for two weeks such as the activity, energy, hair, feces, behavior pattern, etc.
2.5. Evaluation of bone regeneration in vivo 2.5.1. Radiological examination At the predetermined 4 weeks, 12 weeks and 20 weeks post-surgery, three anesthetic rabbits were radiographed using a digital X-ray unit (Siemens, Germany) with a 5 s exposure (62 kVP, 250 mA). All the radiograms were observed at an appropriate magnification, and the grey-scale displayed automatically by the digital X-ray imaging system was applied to estimate the degree of new bone formation. 2.5.2. Micro-computed tomography (micro-CT, mCT) Every three operated New Zealand white rabbits were sacrificed at the predetermined time of 4 weeks, 12 weeks and 20 weeks with an overdose of sodium pentobarbital IV. The fresh intact craniums in airtight cylindrical sample holder were subjected to mCT80 (Scanco Medical AG, Bassersdorf, Switzerland) as described previously [40]. In brief, mCT was performed using an isotropic resolution of 25 mm at the volumes of 55 kVp with an appropriate constrained Gaussian filter (s ¼ 1.2 and width ¼ 1) to partly suppress the noise. The selected volume of interest (VOI) was located in the defect region of the specimen for the subsequent new bone analysis. After scanning the 2-dimensional (2D) slices, 3D images were reconstructed using a cone-beam algorithm provided by Scanco [41]. 2.5.3. Histological analysis The fixed specimens were decalcified by submersion in 10% EDTA (pH 7.0) for 4 weeks at 37 C on a rotating rocker. Then the specimens were serially dehydrated in a graded series of ethanol washes before embedded in paraffin wax. 8 mm thick sections were made and stained according to standard protocols for H&E and Trichrome-Masson. The host response to the implants was determined by observing the inflammatory cells, i.e., macrophage, polymorphonuclear leukocyte, lymphocyte, plasma cell, and giant cell. All histological sections were analyzed with a digital image analysis system (Nikon E 600 Microscope with a Nikon Digital Camera DXM 1200, Nikon Corporation, Japan). The development of the new bone was quantified with a computer-based image analysis system (Image-Pro Plus, Media Cybernetics, Silver Spring, MD, USA) [42]. The percentage of new bone (NB (%)) was expressed as the ratio of the neoformative bone area to the original total defect area as follows: NBð%Þ ¼
neoformative bone area 100% original total defect area
2.5.4. Statistical analysis All data were represented as mean standard deviation with at least three independent sections. Statistical significance of the difference among groups was analyzed by one-way ANOVA. A value of p
Contents lists available at ScienceDirect
Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Injectable thermosensitive PEGePCLePEG hydrogel/acellular bone matrix composite for bone regeneration in cranial defects PeiYan Ni, QiuXia Ding, Min Fan, JinFeng Liao, ZhiYong Qian*, JingCong Luo*, XiuQun Li, Feng Luo, ZhiMing Yang, YuQuan Wei State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, Chengdu, 610041, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 September 2013 Accepted 2 October 2013 Available online 17 October 2013
We have certified that the injectable thermosensitive ABM/PECE composite presented promising potential in bone regeneration benefited from the incorporation of the intrinsic osteoinductive acellular bone matrix (ABM) granules into the poly(ethylene glycol)epoly(ε-capro-lactone)epoly(ethylene glycol) (PEGePCLePEG, PECE) hydrogel. In this study, the 12 mm 8 mm 2 mm cranial defects of the New Zealand white rabbits were fabricated to evaluate the bone regeneration effect. The ABM/PECE composite was injected into the defect while the pure PECE as control, and the bone regeneration was evaluated at 4, 12 and 20 weeks post-surgery by X-radiological examination, micro-computed tomography examination and histological analysis. In ABM/PECE composite treated group, the new bone formed originally from both the margin and the center of the defect, and the defect region had healed up to 20 weeks. Furthermore, the shadow density of the newly formed bone eventually approximated to host cortical bone. In comparison, the control group was filled with sparing new bone with low-density only from the periphery of the defect. Meanwhile, the quantitative determination of new bone by histomorphometry confirmed the excellent bone regeneration of ABM/PECE composite. All the results suggested that the ABM/PECE composite presented enhanced bone regeneration guidance in rabbit cranial defects. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Acellular bone matrix/PEGePCLePEG (ABM/ PECE) Bone regeneration Injectable Thermosensitive hydrogel Cranial defects
1. Introduction The cranial defects resulted from abnormalities, trauma, infection or tumor resection require closure for both functional and esthetic reasons, which have posed great challenge to bone repair materials research and clinical surgeries in the world [1e4]. It is favored to pay close attention to designing and engineering materials of similar composition, structure and properties with the native bone extracellular matrix (ECM) to reconstruct the degenerative or defective cranial bones. During the past several decades, a range of biomaterials have been investigated as bone substitutions. According to the composition, the bone substitutions can be classified mainly into three categories: biological source bone graft, synthesized degradable or undegradable materials and composites of them. The biological source bone graft have been proposed based on their similar composition and structure with the natural ECM, excellent bioactivity, non-cytotoxicity and appropriate strength, * Corresponding authors. Tel.: þ86 28 85164063; fax: þ86 28 85164060. E-mail addresses: [email protected], [email protected] (Z. Qian), [email protected] (J. Luo). 0142-9612/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2013.10.016
including autologous bones [5], allografts [6] and xenografts [7]. However, the application of autologous bones and allografts has been limited in the amount of bone available, potential problems associated with the secondary wound, and difficulties in prefabricating suitable shape and depth to mold the cranial defects cavity [1]. During the past decades, acellular bone matrix (ABM) has attracted increasingly attention as a substitute for recipient bone tissue [8e10]. Besides the advantages described above, ABM with low-immunogenicity has greatly widened source benefited from the acellular operation during the preparation. Therefore, the optimized ABM have been successfully applied in multiple animal models [11e13], and the processed ABM informed the potential to support osteoinduction [14,15]. Furthermore, the ABM can be grounded into granules and prefabricated almost right to mold the cavity even though the bone defects come in a variety of shapes and depths [16,17]. However, the brittle ABM granules might migrate out of the defect cavity and induce severe inflammation, which might bring great pain to the patients [18e20]. One promising strategy to overcome the brittleness of ABM granules is to introduce a optimized polymeric component as a 3D scaffold [21]. Up to now, several biodegradable polymers have been proven to be acceptable in the bone regeneration field to
P. Ni et al. / Biomaterials 35 (2014) 236e248
237
some degree [22,23]. Among them, the injectable hydrogel polymers have been increasingly applicated because of easy administration, minimally invasive procedures, site specific introduction, patient convenience and three-dimensional (3D) network formation under mild conditions [24e27]. In particular, thermosensitive hydrogel could be injected freely at room temperature or lower, and form a gel depot in situ to take the shape of the cavity in the impaired tissue especially at the body temperature. Besides, the thermosensitive hydrogels give a promising modality to avoid using any noxious substances as initiator of phase transition (such as the pH adjustments, oxidants or UV light) owing to the sensitivity to the temperature. Among the intelligent thermosensitive hydrogel, poly(ethylene glycol)poly(ε-capro-lactone)-poly(ethylene glycol) (PEGePCLePEG, PECE) is a linear triblock polyester copolymer composed of hydrophilic poly(ethylene glycol) (PEG) block and hydrophobic poly(ε-capro-lactone) (PCL) block [28e30]. As well known, both PEG and PCL are FDA-approved biomaterials and they have been widely used in biomedical field [31e33]. The physicalechemical properties [34], drug delivery behavior [35], the biodegradability as well as non-cytotoxicity both in vitro and in vivo [30,35] of PECE copolymer have been comprehensively studied in our previous works. In previous works of our group [29], the ABM/PECE hydrogel composite was fabricated by incorporating the bioactive ABM into the biodegradable thermosensitive hydrogel PECE, which could overcome the brittleness of ABM granules. As indicated, ABM/PECE hydrogel composite showed no significant cytotoxicity to rat bone mesenchymal stem cells (MSCs) in vitro and were non-cytotoxic in the BALB/c mice subcutis even up to 4 weeks. Based on the above mentioned investigations, we prospect that the ABM/PECE hydrogel composite could facilitate the bone regeneration in the non-load-bearing cranial repair process by combining the advantages of the intrinsic osteoinductive ABM granules and the injectable thermosensitive PECE hydrogel. The aim of this study was to extend the in vitro results to the in vivo setting by evaluating the effects of ABM/PECE composite in repairing the cranial defects of New Zealand white rabbits. X-ray examination, 3D microcomputed tomography images analysis and histological analysis were performed to evaluate the newly formed bone. 2. Materials and methods 2.1. Materials The (ABM) was prepared with physical and chemical methods as depicted before [36,37]. A section of fresh human femur without soft tissue was soaked in 3% H2O2 to oxidize proteins and reduce antigenicity. After treatment bath, the sectioned bone scaffolds were immersed in the defatting solvent to remove remaining marrow and intramatrix cells. The as-obtained ABM were freeze-dried and ground to granules, the shape and size of which were irregular (1e20 mm). Then the prepared ABM granules were stored in sterile bags at room temperature. Poly(ethylene glycol)poly(ε-capro-lactone)-poly(ethylene glycol) (PEGePCLePEG, PECE) copolymers used in this study were synthesized in two steps as described in previous works briefly [28,29,31]. The number-average molecular weight (Mn) of as-obtained PECE copolymer was 3150, which was determined by the integral intensities of characteristic absorption peaks at 3.60 and 4.07 ppm in the 1H NMR spectrum [38]. After incorporating ABM granules into the PECE hydrogel, the obtained ABM/PECE hydrogel composite (30 wt % ABM) was sterilized by 60Co gamma rays using a set dose of 30 kGy. Other chemical agents like CH2Cl2, petroleum ether, and chloroform were obtained from Chengdu Kelong Chemicals, China. All of them were analytical reagent and used directly without further purification. 2.2. Characterizations of ABM/PECE composite 2.2.1. X-ray diffraction (XRD) X-ray diffraction measurements were used to study the phase analysis and crystallinity of the ABM/PECE composite samples using an X’Pert Pro MPD DY1291 (PHILIPS, Netherlands) diffractometer. The X-ray diffraction was studied using graphite monochromatized Cu K radiation (l ¼ 0.1542 nm; 35 mA; 40 kV) in the 2q ranging from 10 to 60 at a scanning rate of 4 /min.
Fig. 1. X-ray diffraction patterns of pure PECE copolymer (A), ABM granules (B) and ABM/PECE composite (C).
2.2.2. Differential scanning calorimetry (DSC) A differential scanning calorimeter (DSC; NETSCZ 204, NETSCZ, Germany) was used to study the thermal properties of the ABM/PECE hydrogel composite. The dry samples (about 5.0 mg) were loaded in a cell and heat exchange was recorded during the heating and cooling cycle. The samples were firstly heated from room temperature to 100 C under nitrogen atmosphere then cooled to 20 C at a heating rate of 10 C/min, and reheated to 100 C again at the same rate.
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2.2.3. Solegelesol phase transition behavior study Solegelesol phase transition behavior of ABM/PECE hydrogel composite was investigated using test tube-inverting method [34,35]. The sol was defined as ‘flow transparent liquid’ while the gel was defined as ‘no flow opaque solid’, respectively. The solegelesol transition was visually observed by inverting the vials, and the state of the composite held in 1 min. In this study, the hydrogel composite were firstly transited from 10 C to physiological temperature of 37 C, and then back to 10 C. 2.3. Toxicity evaluation in vitro and in vivo 2.3.1. Cytotoxicity, cell proliferation and cell cycle evaluation of ABM/PECE hydrogel composite by WST-1 and flow cytometry The leachates were extracted from ABM/PECE hydrogel composite (30 wt % ABM) for 24 h using DMEM. The sequential dilutions of the stock leachates (100%) were carried out to vary the concentrations (100%, 50%, 25%) [29]. The rat ROS 17/2.8 osteoblasts were incubated with the leachates of different concentrations (0%, 25%, 50%, 100%, n ¼ 3) up to 5 d. The effects of ABM/PECE hydrogel on cytotoxicity and cell proliferation were evaluated by WST-1 method and the absorbance at 570 nm was measured at predetermined 1d, 3d and 5d. Meanwhile, the treated cells were washed twice with PBS and fixed with 70% ethanol after harvest at day 3. The fixed cells were stained with PI solution (50 mg/ml) following RNase A (1 mg/ml) treated, and the cell cycles were analyzed by flow cytometry according to the content of DNA. 2.3.2. Implantation of ABM/PECE hydrogel composite into muscle pouches of rats All the animal experiment protocols were approved by the Institutional Animal Care and Use Committee and were in compliance with all regulatory guidelines. An incision was made at the dorsal sites of the hind legs after the rat was anesthetized with pentobarbital sodium (30 mg/kg, i.p.) and sterilized with iodophor and 70% ethanol solution. The muscle pouches in the dorsal sites were exposed with a tweezer and 0.5 ml of ABM/PECE hydrogel composite was injected. After the operation, the muscle pouch and incision of skin were sewed up respectively and all rats received intramuscular injection of 400,000 units’ penicillin in the following 3 days. The rats were sacrificed at week 2 and week 4 post-surgery, and the implants together with surrounding tissues were harvested and fixed immediately with 10% neutral formalin. Then the samples were embedded in paraffin after a graded dehydration. The sections with a thickness of 5 mm were made by LEICA 2500E diamond saw microtome (Leica SpA, Milan, Italy) and stained according to standard protocols for hematoxylin & eosin (H&E). The sections were analyzed with a digital image analysis system (Nikon E 600 Microscope with a Nikon Digital Camera DXM 1200, Nikon Corporation, Japan). 2.4. Cranial defects construction and bone regeneration evaluation 2.4.1. Surgical procedure To evaluate the bone regeneration, the ABM/PECE hydrogel composite was transplanted into the cranial defects of the New Zealand white rabbits. New Zealand white rabbits (3.0 0.2 kg) used in this study were purchased from the Experimental Animals Center of Sichuan Province, China. All the protocols were approved by the Institutional Animal Care and Use Committee of West China Hospital. Briefly, each New Zealand white rabbits were anaesthetized with pentobarbital sodium (30 mg/kg, i.p.) until the effective anesthesia. The surgical area was shaved and prepared with povidone-iod for aseptic surgery. The calvaria bone was exposed after a straight incision approximately 5 cm in length from the nasal bone to the midsaggital crest, and the periosteum was also incised in the same place. Two rectangular 12 mm 8 mm 2 mm defects were made by a ground section equipped-in a
dental hand piece with copious physiologic saline irrigation as coolant to remove bone debris. The defects were made on each side of the median sagittal suture and care was exercised to avoid injury of the dura [39]. Subsequently, the right calvaria defect was filled with prepared ABM/PECE composite solution while the left defect was filled with PECE hydrogel as control. After placement of the materials, the soft tissues were closed with sutures. All the rabbits received intramuscular injection of 400,000 units’ penicillin per day during the first week post-operation. Meanwhile, their general conditions were observed continuously for two weeks such as the activity, energy, hair, feces, behavior pattern, etc.
2.5. Evaluation of bone regeneration in vivo 2.5.1. Radiological examination At the predetermined 4 weeks, 12 weeks and 20 weeks post-surgery, three anesthetic rabbits were radiographed using a digital X-ray unit (Siemens, Germany) with a 5 s exposure (62 kVP, 250 mA). All the radiograms were observed at an appropriate magnification, and the grey-scale displayed automatically by the digital X-ray imaging system was applied to estimate the degree of new bone formation. 2.5.2. Micro-computed tomography (micro-CT, mCT) Every three operated New Zealand white rabbits were sacrificed at the predetermined time of 4 weeks, 12 weeks and 20 weeks with an overdose of sodium pentobarbital IV. The fresh intact craniums in airtight cylindrical sample holder were subjected to mCT80 (Scanco Medical AG, Bassersdorf, Switzerland) as described previously [40]. In brief, mCT was performed using an isotropic resolution of 25 mm at the volumes of 55 kVp with an appropriate constrained Gaussian filter (s ¼ 1.2 and width ¼ 1) to partly suppress the noise. The selected volume of interest (VOI) was located in the defect region of the specimen for the subsequent new bone analysis. After scanning the 2-dimensional (2D) slices, 3D images were reconstructed using a cone-beam algorithm provided by Scanco [41]. 2.5.3. Histological analysis The fixed specimens were decalcified by submersion in 10% EDTA (pH 7.0) for 4 weeks at 37 C on a rotating rocker. Then the specimens were serially dehydrated in a graded series of ethanol washes before embedded in paraffin wax. 8 mm thick sections were made and stained according to standard protocols for H&E and Trichrome-Masson. The host response to the implants was determined by observing the inflammatory cells, i.e., macrophage, polymorphonuclear leukocyte, lymphocyte, plasma cell, and giant cell. All histological sections were analyzed with a digital image analysis system (Nikon E 600 Microscope with a Nikon Digital Camera DXM 1200, Nikon Corporation, Japan). The development of the new bone was quantified with a computer-based image analysis system (Image-Pro Plus, Media Cybernetics, Silver Spring, MD, USA) [42]. The percentage of new bone (NB (%)) was expressed as the ratio of the neoformative bone area to the original total defect area as follows: NBð%Þ ¼
neoformative bone area 100% original total defect area
2.5.4. Statistical analysis All data were represented as mean standard deviation with at least three independent sections. Statistical significance of the difference among groups was analyzed by one-way ANOVA. A value of p
