Evaluation of oriented electrospun fibers for periosteal flap regeneration in biomimetic triphasic osteochondral implant Xudong Liu,1 Shen Liu,1 Shenghe Liu,1 Wenguo Cui1,2,3* 1
Department of Orthopaedics, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai 200233, People’s Republic of China 2 Orthopedic Institute, Soochow University, Suzhou, Jiangsu 215006, People’s Republic of China 3 Department of Orthopedics, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215006, People’s Republic of China Received 28 September 2013; revised 14 December 2013; accepted 30 January 2014 Published online 20 February 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33119 Abstract: Osteochondral defects represent a serious clinical problem. Although the cell-scaffold complexes have been reported to be effective for repairing osteochondral defects, a periosteal flap is frequently needed to arrest leakage of the implanted cells into the defect and to contribute to the secretion of cytokines to stimulate cartilage repair. The electrospun mesh mimicking the function of the flap assists tissue regeneration by preventing cell leakage and merits favorable outcomes in the cartilaginous region. In this study, an oriented poly(e-caprolactone) (PCL) fibrous membrane (OEM) was fabricated by electrospinning as a periosteal scaffold and then freeze-dried with a collagen type I and hyaluronic acid cartilage scaffold (CH) and finally, freeze-dried with a tricalcium phosphate (TCP) bone substratum. Scanning electron microscopic images show obvious microstructure formation of the trilayered scaffolds, and electrospun fibrous mem-
branes have an oriented fibrous network structure for the periosteal phase. Also shown are opened and interconnected pores with well designed three-dimensional structure, able to be bound in the CH (chondral phase) and TCP (osseous phase) scaffolds. In vitro results showed that the OEM can promote the orientation of bone marrow mesenchymal stem cell (BMSCs) and BMSCs can penetrate into the CH and TCP. After successfully combining the BMSCs, the tissueengineered cartilage which contained the OEM and TCP complex was successfully used to regenerate the osteochondral defects in the rabbit model with greatly improved repair C 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part B: effects. V Appl Biomater, 102B: 1407–1414, 2014.
Key Words: osteochondral defect, electrospinning, oriented fibers, triphasic scaffold, periosteal flap
How to cite this article: Liu X, Liu S, Liu S, Cui W. 2014. Evaluation of oriented electrospun fibers for periosteal flap regeneration in biomimetic triphasic osteochondral implant. J Biomed Mater Res Part B 2014:102B:1407–1414.
INTRODUCTION
As a result of trauma or degenerative alterations of articular cartilage and underlying subchondral bone, osteochondral defects represent a serious clinical problem. This is because it is generally accepted that such combined defects cannot heal spontaneously. The outcome is that the osteochondral defects lead to progressive destruction of a functional, weight-bearing joint.1 Nowadays, although the engineered osteochondral complexes that consisted of cell and scaffold have shown promising results in the repair of damage; and the hierarchical architecture of the articular cartilage and the underlying subchondral bone proves the creation of a substitute a daunting challenge.2–5 The transplantation of autologous osteochondral plugs have been shown to reestablish destroyed bone and cartilage simultaneously.6 However, sources of transplants are
limited and donor site defects have to be considered as new clinical symptoms.7,8 Autologous cell implantation at the defect site can also work. Pregnant with the cells or not, the biphasic osteochondral scaffolds with a chondral phase and an osseous phase have been reported effective in the repair of osteochondral defects.9–11 However, a periosteal flap is frequently needed to arrest leakage of the implanted cells into the defect and to secrete cytokines to stimulate cartilage repair.12,13 Furthermore, as the study of Kajitani et al. showed, there was no significant difference in the outcome of the cartilage defects between the defects that were resurfaced with fresh periosteum grafts and the defects that were resurfaced with frozen periosteum grafts.14 Thereafter, the electrospun mesh can assist tissue regeneration by preventing cell leakage and merit favorable outcomes in the region of the cartilage.
Correspondence to: W. Cui (e-mail: [email protected]) Contract grant sponsor: Shanghai Municipal Commission of Science and Technology Program Rising-Star Program; contract grant number: 13QH1401900 Contract grant sponsor: National Natural Science Foundation of China; contract grant number: 51373112, 81301545, and 30700453
C 2014 WILEY PERIODICALS, INC. V
1407
Recently, oriented electrospun fibrous membrane has been introduced to induce chondrogenic differentiation of human mesenchymal stem cells and fiber-guided cell orientation in the superficial zone of articular cartilage.15 In this study, an oriented poly(e-caprolactone) (PCL) electrospun fibrous membrane (OEM) was fabricated and then freezedried with a collagen type I (Col-I) and hyaluronic acid (HA) cartilage scaffold (CH). Subsequently, the combined CH was freeze-dried with a tricalcium phosphate (TCP) bone substratum. Bone marrow mesenchymal stem cells (BMSCs) were used as seed cells. It was hypothesized that the OEM/ CH/TCP scaffold could benefit the reconstruction of osteochondral defects without suture. MATERIALS AND METHODS
Materials PCL (Mw 5 50 kDa, Mw/Mn 5 1.6) was prepared by bulk ring-opening polymerization (Jinan Daigang Co., Jinan, China). Col-I was purchased from Sichuan Ming-Rang BioTech Co. Ltd, China, and HA (sodium salt, Mw 5 0.5 MDa) was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Porous TCP cylinders (diameter 5 mm, height 5 mm) were purchased from Bio-Lu (Shanghai Bio-Lu Co., Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum were purchased from Gibco (Grand Island, NY). All other reagents and media were of reagent grade or better and were purchased from Invitrogen, unless otherwise indicated.
Fabrication of scaffolds PCL electrospun solution could be obtained by dissolving 1 g of PCL in 4.5 g of chloroform solvent. The electrospinning processes were performed as described previously.16 A grounded, rolling pole drum with a diameter of 8 cm was used as a collector, and the rotational speed was set to 3000 rpm. Then, the oriented PCL electrospun fibrous membrane was fabricated by an electrospinning technique at a voltage of 15 kV and then fed at a feed rate of 3 mL/h. The OEM was collected on the surface of the high-speed rolling drum and vacuum-dried at room temperature for 24 h. The Col-I and HA CHs were fabricated via freeze-drying. The Col-I and HA were dissolved in 0.05M acetic acid under consistent, overnight stirring, to yield the blended solution with a concentration of 1% (Col-I:HA 5 1:1, w/w). The ColI/HA solution was subsequently frozen at 280 C for 6 h and then freeze-dried overnight to obtain the chondral phrase. Freeze-dried Col-I/HA CHs were further crosslinked with 50 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to obtain stable porous scaffolds.17 To integrate the OEM and CH into the osseous phase (TCP), OEM and TCP were first pre-wet with Col-I/HA solution (dissolved in 0.05M acetic acid) (1 wt %, ColI:HA 5 1:1, w/w) for 6 h. Thereafter, the rounded CH was placed on the top of the TCP while the membrane was placed on the top of the CH. The combined scaffolds were frozen at 280 C for 6 h followed by overnight freeze-drying and were crosslinked using EDC.
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Characteristics of the scaffolds Surface morphology. The morphology of all specimens was observed by scanning electron microscopy (SEM, FEI Quanta 200, The Netherlands). For observation, all specimens were sputter coated with gold. The surface morphology was determined at an accelerated voltage of 10 kV. Porosity. Porosities of the chondral phase and the osseous phase of the triphasic scaffold were determined using the method of liquid displacement.18 Briefly, the scaffold was immersed in a given volume (V1) of hexane and the total volume of the scaffold and hexane was taken as V2. The resulting volume was recorded as V3, after removing the scaffold. The porosity of the chondral phase and osseous phase scaffolds were calculated by (V1 2 V3)/(V2 2 V3). Mechanical testing. Uniaxial tensile tests for PCL fibrous membranes (70 mm 3 7 mm) were performed using an allpurpose mechanical testing machine, moving at a speed of 0.5 mm/s (Instron 5567, Norwood, MA) in the directions parallel to the fibrous orientation of the scaffolds. Stress– strain testing of the chondral phase (30.0 mm 3 10.0 mm 3 10.0 mm) was performed for compressive strength using a universal material tester (H5K-S, Hounsfield, UK) with a load cell of 50 N at a cross-head speed of 10 mm/min (n 5 5). Compressive strength of cylindrical TCP specimens was tested using a mechanical testing machine (5500R100kN, Instron, USA) at a load cell of 1000 N and a cross speed of 10 mm/min (n 5 5). Isolation and culture of rabbit MSCs Animal experiments were carried out in accordance with the policies of the National Institutes of Health of People’s Republic of China. BMSCs were obtained from 16-week-old New Zealand white rabbits and further cultured. Briefly, after intramuscular administration of ketamine hydrochloride (60 mg/kg) and xylazine (6 mg/kg), about 5 mL of marrow was extracted from the right iliac crest, using a needle. Then, the marrow was flushed with 10 mL DMEM supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 mg/mL streptomycin, and cultured in a humidified incubator (37 C, 5% CO2). The red cells were removed via the culture medium changing after 5 days. Third-passage cells were used for further study. Gross and microscopic analyses of in vitro-cultured trilayered scaffold To observe cytoskeletal arrangements, BMSCs were pipetted directly onto the OEM at a density of 3 3 104 cells/cm2. Before cell seeding, the OEMs were sterilized by immersion in 75% ethanol for 1.5 h. After 24 h cultures, the BMSCs on the OEMs were fixed in 4% paraformaldehyde for 10 min. After removing the fixative, the cells were rinsed three times in phosphate buffer saline (PBS). They were then permeabilized using 0.2% Triton X-100 (Sigma Aldrich) for 10 min, and then again rinsed three times in PBS. The cytoskeleton was stained with 20 lg/mL of phalloidin (Sigma) for 1 h and the cell nuclei were stained with 1 lg/mL DAPI for 5
EVALUATION OF ORIENTED ELECTROSPUN FIBERS
ORIGINAL RESEARCH REPORT
min, respectively. Then, a confocal laser scanning microscope (Leica TCS SP2; Leica Microsystems, Heidelberg, Germany) was used for observation. To detect the BMSCs/CH and BMSCs/TCP scaffold composites, both composites were pre-immersed in culture media for 24 h to promote cell attachment. The cells were directly pipetted into both composites at a final seeding density of 3 3 106 cells/scaffold, and then cell-scaffold constructs were incubated for 4 h to allow the cells to completely adhere to the scaffolds. Then the constructs were placed in a six-well plate and incubated in a culture medium. After incubation for 21 days, the BMSCs/CH was fixed in 4% paraformaldehyde. Five micrometer cross sections were obtained after dehydration, clarification, infiltration, and paraffin embedding. The sections were stained with Hematoxylin and eosin (H&E) and safranin O, and images were obtained using a microscope (LEICA DM 4000 B). Both chondral and osseous phases were observed by SEM (FEI Quanta 200, The Netherlands) after fixation with 2.5% glutaraldehyde (Gibco Laboratories) and subsequent dehydration through a graded ethanol series. In vivo experimental designs and surgical procedures The cell-scaffold constructs were prepared as the in vitro study. The scaffolds were implanted in the osteochondral defect within the knees of the rabbits. After anesthetized, as described in the previous section, a 5-cm medial parapatellar incision was made to explore the patellofemoral joint. An osteochondral defect (diameter, 5 mm; depth, 6 mm) was created in the patellar groove of the distal femur. Then, the scaffold was inserted into the defect for press-fit fixation until the chondral phase was parallel with the articular surface of the tibia. The rabbits were divided into four groups (n 5 10, each group): BMSCs/OEM/CH/TCP composite (group I); BMSCs/CH/TCP composite (group II); osteochondral autograft (group III); and untreated (group IV). Gross evaluation of regenerated tissue The animals were euthanized by an overdose of anesthesia at 12 weeks after surgery. After opening the articular capsules, each joint was photographed and examined, utilizing the International Cartilage Repair Society (ICRS) Macroscopic Score to evaluate the degree of defect repair, integration of border zone, and macroscopic appearance.19 Histological and immunohistochemical analyses After gross examination, the retrieved knees were fixed in 4% paraformaldehyde overnight and decalcified in 10% EDTA for 1 month, at room temperature. Then, samples were dehydrated by graded ethanol, followed by paraffin embedding. Samples were cut into 4-lm slices and stained with toluidine blue and safranin O/fast green. The repaired tissues were graded blindly by two observers for the evaluation of hyaline cartilage formation, structural characteristics, and tissue morphology in the defects, using the ICRS Visual Histological Assessment Scale.19 The expression of Col-I and Col-II was analyzed by immunohistological staining. The sections were dewaxed in
xylene and then hydrated through decreasing concentrations of alcohol. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide. After subsequent blocking with goat serum (Sigma) (1:100 dilution), the sections were incubated with primary antibodies of collagen II (Neomarkers) or collagen I (Abcam) overnight at 4 C. They were then washed three times in PBS, and then incubated with secondary antibodies for 1 h at 37 C. Staining was developed in DAB solution (Dako, Hamburg, Germany), with counterstaining by hematoxylin. Mechanical evaluation The compressive mechanical properties of the joint surface were tested with an Instron testing machine (model 5543; Instron) using a 2-mm diameter cylindrical indenter fitted with a 10 N maximum loading cell as a previous study.20 The unconfined equilibrium modulus was determined by applying a step displacement (20% strain) and monitoring compressive force applied until equilibrium was reached. The crosshead speed used was approximately 0.06 mm/ min. The ratio of equilibrium force applied to the crosssectional area was divided by the applied strain to calculate the equilibrium modulus. Statistical analysis Descriptive statistics were used to determine group means and standard deviations. The analysis was performed using analysis of variance (ANOVA) for multiple comparisons. Statistical significance was defined as a p-value
Department of Orthopaedics, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai 200233, People’s Republic of China 2 Orthopedic Institute, Soochow University, Suzhou, Jiangsu 215006, People’s Republic of China 3 Department of Orthopedics, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215006, People’s Republic of China Received 28 September 2013; revised 14 December 2013; accepted 30 January 2014 Published online 20 February 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33119 Abstract: Osteochondral defects represent a serious clinical problem. Although the cell-scaffold complexes have been reported to be effective for repairing osteochondral defects, a periosteal flap is frequently needed to arrest leakage of the implanted cells into the defect and to contribute to the secretion of cytokines to stimulate cartilage repair. The electrospun mesh mimicking the function of the flap assists tissue regeneration by preventing cell leakage and merits favorable outcomes in the cartilaginous region. In this study, an oriented poly(e-caprolactone) (PCL) fibrous membrane (OEM) was fabricated by electrospinning as a periosteal scaffold and then freeze-dried with a collagen type I and hyaluronic acid cartilage scaffold (CH) and finally, freeze-dried with a tricalcium phosphate (TCP) bone substratum. Scanning electron microscopic images show obvious microstructure formation of the trilayered scaffolds, and electrospun fibrous mem-
branes have an oriented fibrous network structure for the periosteal phase. Also shown are opened and interconnected pores with well designed three-dimensional structure, able to be bound in the CH (chondral phase) and TCP (osseous phase) scaffolds. In vitro results showed that the OEM can promote the orientation of bone marrow mesenchymal stem cell (BMSCs) and BMSCs can penetrate into the CH and TCP. After successfully combining the BMSCs, the tissueengineered cartilage which contained the OEM and TCP complex was successfully used to regenerate the osteochondral defects in the rabbit model with greatly improved repair C 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part B: effects. V Appl Biomater, 102B: 1407–1414, 2014.
Key Words: osteochondral defect, electrospinning, oriented fibers, triphasic scaffold, periosteal flap
How to cite this article: Liu X, Liu S, Liu S, Cui W. 2014. Evaluation of oriented electrospun fibers for periosteal flap regeneration in biomimetic triphasic osteochondral implant. J Biomed Mater Res Part B 2014:102B:1407–1414.
INTRODUCTION
As a result of trauma or degenerative alterations of articular cartilage and underlying subchondral bone, osteochondral defects represent a serious clinical problem. This is because it is generally accepted that such combined defects cannot heal spontaneously. The outcome is that the osteochondral defects lead to progressive destruction of a functional, weight-bearing joint.1 Nowadays, although the engineered osteochondral complexes that consisted of cell and scaffold have shown promising results in the repair of damage; and the hierarchical architecture of the articular cartilage and the underlying subchondral bone proves the creation of a substitute a daunting challenge.2–5 The transplantation of autologous osteochondral plugs have been shown to reestablish destroyed bone and cartilage simultaneously.6 However, sources of transplants are
limited and donor site defects have to be considered as new clinical symptoms.7,8 Autologous cell implantation at the defect site can also work. Pregnant with the cells or not, the biphasic osteochondral scaffolds with a chondral phase and an osseous phase have been reported effective in the repair of osteochondral defects.9–11 However, a periosteal flap is frequently needed to arrest leakage of the implanted cells into the defect and to secrete cytokines to stimulate cartilage repair.12,13 Furthermore, as the study of Kajitani et al. showed, there was no significant difference in the outcome of the cartilage defects between the defects that were resurfaced with fresh periosteum grafts and the defects that were resurfaced with frozen periosteum grafts.14 Thereafter, the electrospun mesh can assist tissue regeneration by preventing cell leakage and merit favorable outcomes in the region of the cartilage.
Correspondence to: W. Cui (e-mail: [email protected]) Contract grant sponsor: Shanghai Municipal Commission of Science and Technology Program Rising-Star Program; contract grant number: 13QH1401900 Contract grant sponsor: National Natural Science Foundation of China; contract grant number: 51373112, 81301545, and 30700453
C 2014 WILEY PERIODICALS, INC. V
1407
Recently, oriented electrospun fibrous membrane has been introduced to induce chondrogenic differentiation of human mesenchymal stem cells and fiber-guided cell orientation in the superficial zone of articular cartilage.15 In this study, an oriented poly(e-caprolactone) (PCL) electrospun fibrous membrane (OEM) was fabricated and then freezedried with a collagen type I (Col-I) and hyaluronic acid (HA) cartilage scaffold (CH). Subsequently, the combined CH was freeze-dried with a tricalcium phosphate (TCP) bone substratum. Bone marrow mesenchymal stem cells (BMSCs) were used as seed cells. It was hypothesized that the OEM/ CH/TCP scaffold could benefit the reconstruction of osteochondral defects without suture. MATERIALS AND METHODS
Materials PCL (Mw 5 50 kDa, Mw/Mn 5 1.6) was prepared by bulk ring-opening polymerization (Jinan Daigang Co., Jinan, China). Col-I was purchased from Sichuan Ming-Rang BioTech Co. Ltd, China, and HA (sodium salt, Mw 5 0.5 MDa) was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Porous TCP cylinders (diameter 5 mm, height 5 mm) were purchased from Bio-Lu (Shanghai Bio-Lu Co., Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum were purchased from Gibco (Grand Island, NY). All other reagents and media were of reagent grade or better and were purchased from Invitrogen, unless otherwise indicated.
Fabrication of scaffolds PCL electrospun solution could be obtained by dissolving 1 g of PCL in 4.5 g of chloroform solvent. The electrospinning processes were performed as described previously.16 A grounded, rolling pole drum with a diameter of 8 cm was used as a collector, and the rotational speed was set to 3000 rpm. Then, the oriented PCL electrospun fibrous membrane was fabricated by an electrospinning technique at a voltage of 15 kV and then fed at a feed rate of 3 mL/h. The OEM was collected on the surface of the high-speed rolling drum and vacuum-dried at room temperature for 24 h. The Col-I and HA CHs were fabricated via freeze-drying. The Col-I and HA were dissolved in 0.05M acetic acid under consistent, overnight stirring, to yield the blended solution with a concentration of 1% (Col-I:HA 5 1:1, w/w). The ColI/HA solution was subsequently frozen at 280 C for 6 h and then freeze-dried overnight to obtain the chondral phrase. Freeze-dried Col-I/HA CHs were further crosslinked with 50 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to obtain stable porous scaffolds.17 To integrate the OEM and CH into the osseous phase (TCP), OEM and TCP were first pre-wet with Col-I/HA solution (dissolved in 0.05M acetic acid) (1 wt %, ColI:HA 5 1:1, w/w) for 6 h. Thereafter, the rounded CH was placed on the top of the TCP while the membrane was placed on the top of the CH. The combined scaffolds were frozen at 280 C for 6 h followed by overnight freeze-drying and were crosslinked using EDC.
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Characteristics of the scaffolds Surface morphology. The morphology of all specimens was observed by scanning electron microscopy (SEM, FEI Quanta 200, The Netherlands). For observation, all specimens were sputter coated with gold. The surface morphology was determined at an accelerated voltage of 10 kV. Porosity. Porosities of the chondral phase and the osseous phase of the triphasic scaffold were determined using the method of liquid displacement.18 Briefly, the scaffold was immersed in a given volume (V1) of hexane and the total volume of the scaffold and hexane was taken as V2. The resulting volume was recorded as V3, after removing the scaffold. The porosity of the chondral phase and osseous phase scaffolds were calculated by (V1 2 V3)/(V2 2 V3). Mechanical testing. Uniaxial tensile tests for PCL fibrous membranes (70 mm 3 7 mm) were performed using an allpurpose mechanical testing machine, moving at a speed of 0.5 mm/s (Instron 5567, Norwood, MA) in the directions parallel to the fibrous orientation of the scaffolds. Stress– strain testing of the chondral phase (30.0 mm 3 10.0 mm 3 10.0 mm) was performed for compressive strength using a universal material tester (H5K-S, Hounsfield, UK) with a load cell of 50 N at a cross-head speed of 10 mm/min (n 5 5). Compressive strength of cylindrical TCP specimens was tested using a mechanical testing machine (5500R100kN, Instron, USA) at a load cell of 1000 N and a cross speed of 10 mm/min (n 5 5). Isolation and culture of rabbit MSCs Animal experiments were carried out in accordance with the policies of the National Institutes of Health of People’s Republic of China. BMSCs were obtained from 16-week-old New Zealand white rabbits and further cultured. Briefly, after intramuscular administration of ketamine hydrochloride (60 mg/kg) and xylazine (6 mg/kg), about 5 mL of marrow was extracted from the right iliac crest, using a needle. Then, the marrow was flushed with 10 mL DMEM supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 mg/mL streptomycin, and cultured in a humidified incubator (37 C, 5% CO2). The red cells were removed via the culture medium changing after 5 days. Third-passage cells were used for further study. Gross and microscopic analyses of in vitro-cultured trilayered scaffold To observe cytoskeletal arrangements, BMSCs were pipetted directly onto the OEM at a density of 3 3 104 cells/cm2. Before cell seeding, the OEMs were sterilized by immersion in 75% ethanol for 1.5 h. After 24 h cultures, the BMSCs on the OEMs were fixed in 4% paraformaldehyde for 10 min. After removing the fixative, the cells were rinsed three times in phosphate buffer saline (PBS). They were then permeabilized using 0.2% Triton X-100 (Sigma Aldrich) for 10 min, and then again rinsed three times in PBS. The cytoskeleton was stained with 20 lg/mL of phalloidin (Sigma) for 1 h and the cell nuclei were stained with 1 lg/mL DAPI for 5
EVALUATION OF ORIENTED ELECTROSPUN FIBERS
ORIGINAL RESEARCH REPORT
min, respectively. Then, a confocal laser scanning microscope (Leica TCS SP2; Leica Microsystems, Heidelberg, Germany) was used for observation. To detect the BMSCs/CH and BMSCs/TCP scaffold composites, both composites were pre-immersed in culture media for 24 h to promote cell attachment. The cells were directly pipetted into both composites at a final seeding density of 3 3 106 cells/scaffold, and then cell-scaffold constructs were incubated for 4 h to allow the cells to completely adhere to the scaffolds. Then the constructs were placed in a six-well plate and incubated in a culture medium. After incubation for 21 days, the BMSCs/CH was fixed in 4% paraformaldehyde. Five micrometer cross sections were obtained after dehydration, clarification, infiltration, and paraffin embedding. The sections were stained with Hematoxylin and eosin (H&E) and safranin O, and images were obtained using a microscope (LEICA DM 4000 B). Both chondral and osseous phases were observed by SEM (FEI Quanta 200, The Netherlands) after fixation with 2.5% glutaraldehyde (Gibco Laboratories) and subsequent dehydration through a graded ethanol series. In vivo experimental designs and surgical procedures The cell-scaffold constructs were prepared as the in vitro study. The scaffolds were implanted in the osteochondral defect within the knees of the rabbits. After anesthetized, as described in the previous section, a 5-cm medial parapatellar incision was made to explore the patellofemoral joint. An osteochondral defect (diameter, 5 mm; depth, 6 mm) was created in the patellar groove of the distal femur. Then, the scaffold was inserted into the defect for press-fit fixation until the chondral phase was parallel with the articular surface of the tibia. The rabbits were divided into four groups (n 5 10, each group): BMSCs/OEM/CH/TCP composite (group I); BMSCs/CH/TCP composite (group II); osteochondral autograft (group III); and untreated (group IV). Gross evaluation of regenerated tissue The animals were euthanized by an overdose of anesthesia at 12 weeks after surgery. After opening the articular capsules, each joint was photographed and examined, utilizing the International Cartilage Repair Society (ICRS) Macroscopic Score to evaluate the degree of defect repair, integration of border zone, and macroscopic appearance.19 Histological and immunohistochemical analyses After gross examination, the retrieved knees were fixed in 4% paraformaldehyde overnight and decalcified in 10% EDTA for 1 month, at room temperature. Then, samples were dehydrated by graded ethanol, followed by paraffin embedding. Samples were cut into 4-lm slices and stained with toluidine blue and safranin O/fast green. The repaired tissues were graded blindly by two observers for the evaluation of hyaline cartilage formation, structural characteristics, and tissue morphology in the defects, using the ICRS Visual Histological Assessment Scale.19 The expression of Col-I and Col-II was analyzed by immunohistological staining. The sections were dewaxed in
xylene and then hydrated through decreasing concentrations of alcohol. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide. After subsequent blocking with goat serum (Sigma) (1:100 dilution), the sections were incubated with primary antibodies of collagen II (Neomarkers) or collagen I (Abcam) overnight at 4 C. They were then washed three times in PBS, and then incubated with secondary antibodies for 1 h at 37 C. Staining was developed in DAB solution (Dako, Hamburg, Germany), with counterstaining by hematoxylin. Mechanical evaluation The compressive mechanical properties of the joint surface were tested with an Instron testing machine (model 5543; Instron) using a 2-mm diameter cylindrical indenter fitted with a 10 N maximum loading cell as a previous study.20 The unconfined equilibrium modulus was determined by applying a step displacement (20% strain) and monitoring compressive force applied until equilibrium was reached. The crosshead speed used was approximately 0.06 mm/ min. The ratio of equilibrium force applied to the crosssectional area was divided by the applied strain to calculate the equilibrium modulus. Statistical analysis Descriptive statistics were used to determine group means and standard deviations. The analysis was performed using analysis of variance (ANOVA) for multiple comparisons. Statistical significance was defined as a p-value
