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Radially oriented collagen scaffold with SDF-1 promotes osteochondral repair by facilitating cell homing
Biomaterials 39 (2015) 114e123
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Radially oriented collagen scaffold with SDF-1 promotes osteochondral repair by facilitating cell homing Pengfei Chen a, b, 1, Jiadong Tao a, 1, Shouan Zhu a, 1, Youzhi Cai a, Qijiang Mao a, Dongsheng Yu a, Jun Dai a, HongWei Ouyang a, * a
Center for Stem Cell and Tissue Engineering, School of Medicine, Zhejiang Provincial Key Lab for Tissue Engineering and Regenerative Medicine, Zhejiang University, 866 Yu Hang Tang Road, Hangzhou 310058, China Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, 3 East Qingchun Road, Hangzhou 310016, China
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 July 2014 Accepted 19 October 2014 Available online
The migration of cells from the side and the bottom of the defect is important in osteochondral defect healing. Here, we designed a novel collagen scaffold that possessed channels in both the horizontal and the vertical directions, along with stromal cell-derived factor-1 (SDF-1) to enhance osteochondral regeneration by facilitating cell homing. Firstly we fabricated the radially oriented and random collagen scaffolds, then tested their properties. The radially oriented collagen scaffold had better mechanical properties than the random scaffold, but both supported cell proliferation well. Then we measured the migration of BMSCs in the scaffolds in vitro. The radially oriented collagen scaffold effectively promoted their migration, and this effect was further facilitated by addition of SDF-1. Moreover, we created osteochondral defects in rabbits, and implanted them with random or oriented collagen scaffolds with or without SDF-1, and evaluated cartilage and subchondral bone regeneration at 6 and 12 weeks after surgery. Cartilage regeneration with both the radially oriented scaffold and SDF-1 effectively promoted repair of the cartilage defect. Our results confirmed that the implantation of the radially oriented channel collagen scaffold with SDF-1 could be a promising strategy for osteochondral repair. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Osteochondral tissue engineering Cartilage defect repair SDF-1 Collagen scaffolds
1. Introduction Tissue or organ defect is the final clinical manifestation of all refractory diseases. For decades, cell-based methods have been used to substitute pathologic cells and to cure tissue defects [15]. However, cell delivery has shortcomings such as immune rejection, pathogen transmission, potential tumorigenesis, issues with packaging, storage, and difficulties in clinical adoption and regulatory approval [68]. Osteoarthritis (OA), the most common form of arthritis, is a group of mechanical abnormalities resulting in the articular cartilage degradation [9]. Though cell transplantation for cartilage regeneration is well studied [1013], defect regeneration by cell homing, especially without cell transplantation, remains a promising approach lacking experimental testing [1416].
* Corresponding author. Mailbox #39, Center for Stem Cell and Tissue Engineering, School of Medicine, Zhejiang University, #866 Yu Hang Tang Road, Hangzhou 310058, China. Tel./fax: þ86 571 88208262. E-mail address: [email protected] (H. Ouyang). 1 Authors contribute equally to this work. http://dx.doi.org/10.1016/j.biomaterials.2014.10.049 0142-9612/© 2014 Elsevier Ltd. All rights reserved.
The predominant model of cartilage regeneration is focal lesions such as osteochondral defects [17]. It is generally accepted that osteochondral defect healing is developed from the bottom of the defect where bone marrow-derived MSCs (BMSCs) contribute to the regeneration. However, previous serial cartilage studies suggest that during cartilage repair segmental neo-cartilage is formed by adjacent tissue protruding [1820]. These findings suggest that the migration of cells from the sides of defect is also important in osteochondral defect healing. Thus the choices of an appropriate scaffold which can facilitate cell migration are of great importance. The utilization of biphasic and multiphasic scaffolds to replicate the multiphasic nature of the native tissue is the core challenge and precondition for the organization of living cells to functional tissue. Designing scaffolds with tissue-specific architecture is an effective way for osteochondral repair [2123]. Meanwhile the threedimensional environment of the scaffolds such as pore size and orientation will affect the extracellular matrix deposition, as cells infiltrate into the scaffold will align with the pore and follow the channels arranged by the scaffold [24]. So it seems that current popular osteochondral scaffolds with random structures cannot facilitate the process of cell homing. There is, therefore, a critical
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need to modify the structural properties of scaffolds in a radially oriented manner to achieve satisfactory outcomes in cartilage repair. Here, we have designed a novel radially oriented collagen scaffold, which has oriented channels in both the horizontal and the vertical directions. We further assessed the therapeutic effects of this biological material for osteochondral repair. SDF-1, the unique ligand of its specific receptor, chemokine (CX-C motif) receptor 4 (CXCR-4), is constitutively expressed in many organs [25]. Studies have demonstrated that SDF-1 stimulates stem-cell homing [26] and migration [25]. Bone marrow stromal cells (BMSCs), express both SDF-1 and CXCR-4 and, hence, BMSCs promote their own survival and proliferation through an autocrine or paracrine mechanism [27]. The general approach for SDF-1 delivery in tissue engineering is direct protein incorporation into scaffolds which has succeeded in improving the regeneration of tissue or organ defect [28,29]. Our previous studies also demonstrated that SDF-1 is one of the key factors for MSC migration in the subchondral bone environment and can be used osteochondral repair [19,30]. In this study, we hypothesized that our radially oriented collagen scaffold, along with SDF-1, promoted osteochondral repair by facilitating cell homing. To test this hypothesis, experiments
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were designed as follows: (1) fabrication and characterization of a radially oriented channel collagen scaffold; (2) in vitro testing of the efficiency of this scaffold along with SDF-1 in promoting MSC migration; and (3) in vivo analysis of the effect of this scaffold with SDF-1 on the healing of osteochondral defects in rabbits. 2. Materials and methods 2.1. Materials Jinhua pigs (<100 kg, <6-month-old) of either gender were used as donors. Type I collagen was isolated and purified from pig tendon in our lab. Reagents were from Sigma unless specifically indicated. 2.2. Fabrication of the radially oriented and random collagen scaffold The radially oriented scaffold was made using a temperature gradient-guided thermal-induced phase-separation technique (Fig. 1a). The pig tendons were dissected out, sliced and washed with several changes of cold distilled water to remove plasma proteins, and then extracted with 1 M NaCl in 50 mM TriseHCl (pH 7.4) and constantly stirred overnight at 4 C. The supernatant was decanted and the remainder was washed with several changes of cold distilled water to remove salts. Then the tendon was incubated in 0.5 M acetic acid (1.0% w/v) with 1 mg/ml pepsin overnight at 4 C to obtain an aqueous extract. The extract was precipitated with saline (0.9 M NaCl) and dissolved in 0.5 M HAc. The solution was then dialyzed, frozen and freeze-dried to obtain the collagen. The collagen was dissolved again in 0.05 M acetic acid to form 10 mg/ml solution. Then the collagen solution was added to a
Fig. 1. Macroscopic and microscopic structures of the collagen scaffolds. (a) Fabrication of the radially oriented and random collagen scaffolds. (b) Microscopic structure of the collagen scaffolds.
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mold with an inner diameter of 5.0 mm and height of 20.0 mm. The wall of the mold was made of copper, which was at a temperature of 20 C as a cooling source. In the middle of the mold, there was a plastic rod 1.5 mm in diameter. The top and bottom of the mold was also made of plastic. Both the top and bottom were designed as thermal insulation material. With evaporation of the liquid nitrogen, the collagen solution was unidirectionally frozen from the edge to the center of the solution. The radially oriented collagen crystallization occurred in half an hour. For the random scaffold, the collagen solution was added into a mold of the same size. But the top and bottom were made of copper. After addition of the collagen solution, the mold was put into liquid nitrogen at 196 C for 10 min. Then random collagen crystallization occurred. Next, both the radially oriented and randomly crystallized collagen were kept at 80 C for >4 h. The crystallized materials were removed from the molds and lyophilized in a freeze-dryer under vacuum for 48 h. Then the scaffolds were cut into cylinders 5.0 mm high and subjected to thermal crosslinking, then exposed to 60Co radiation for sterilization before cell culture and in vivo experimentation. Briefly, the thermal crosslinking was carried out at 25 C for 24 h, 110 C for 72 h, and 65 C for 48 h.
2.8. Animal model All animals were treated according to the standard guidelines approved by Zhejiang University Ethics Committee (ZJU 2010105003). 36 adult male New Zealand white rabbits (2.5e3 kg) were maintained singly in stainless-steel cages. After intramuscular and intravenous injection with 10% chloral hydrate (4 ml kg1), the knee joint was opened using a medial parapatellar approach. The patella was dislocated laterally and the surface of the femoropatellar groove exposed. A cylindrical osteochondral defect 4 mm in diameter and 3.5e4 mm deep was created in the patellar groove using a stainless-steel punch. The defect in the left joint was treated with a random scaffold or a radially oriented scaffold, and the right joint was treated with a random scaffold with SDF-1 or a radially oriented scaffold with SDF-1 (n ¼ 14 joints in each group). 8 rabbits were treated with sham operations as controls. The joint capsule and skin were closed with interrupted sutures. Immediately after surgery, the animals were returned to their individual cages without joint immobilization. A postoperative antibiotic (gentamicin) was administered intramuscularly at 400,000 U per day for 3 days. After sacrifice at 6 and 12 weeks, knee joints from each group were evaluated histologically.
2.3. Characterization of the radially oriented and random collagen scaffolds The collagen scaffold samples were cut in half in both vertical and cross-section, mounted on aluminum stubs, sputter-coated with gold, and observed under a scanning electron microscope (Hitachi S3000N) at an accelerating voltage of 15 kV. The mechanical properties of the scaffolds were tested using an Instron testing machine (model 5543; Instron, Canton, MA) and software (Bluehill V2.0; Instron). It was a compression testing at a speed of 2.0 mm/min (without the tare load). According to the normal range of deformation, the deformation range of 0%e10% was chosen. To show the change of the stress with different compression degrees, the scaffold with a height of 5.0 ± 0.2 mm was placed horizontally between two mechanical detecting heads during the testing. 12 samples from the random and radially oriented scaffolds were tested. 2.4. Isolation and culture of bone marrow mesenchymal stem cells BMSCs were isolated by short-term adherence to plastic as described previously [31]. Rabbit bone marrow was aspirated from the iliac crest of 4-month-old female New Zealand white rabbits. Nucleated cells were isolated by centrifugation at 1200 rpm for 5 min and the supernatant was removed. Following this, the nucleated cell layer was carefully collected and re-suspended in culture medium containing Dulbecco's modified Eagle's medium (DMEM), 10% w/v fetal bovine serum (FBS), 100 units/ml penicillin, and 100 mg/ml streptomycin (all from Gibco). Cells were plated at 5 106 per 100 mm dish and incubated at 37 C under 5% humidified CO2. After 48 h, non-adherent cells were discarded and adherent cells were continuously cultured. The medium was changed every 3 days. When cells became nearly confluent, they were detached with trypsin and serially sub-cultured. 2.5. Cell viability assay A 0.4 cm diameter collagen membrane was immersed in 0.12 ml DMEM-high glucose containing 10% FBS and 1% penicillin/streptomycin for >24 h. BMSCs were seeded on a 96-well plate at 103 cells/well. After 24 h, the medium of the test group was replaced with leaching liquor acquired from collagen membrane or fresh DMEM-high glucose containing 10% FBS and 1% penicillin/streptomycin or 5% DMSO. After 3 days, the cytotoxicity of the collagen membrane was assessed using Cell Counting KIT-8 (CCK-8, Dojindo, Kumamoto, Japan) at 450 nm. Cell number was correlated with optical density (OD). 2.6. Cell proliferation assay Cell proliferation was measured using CCK-8. The BMSC-seeded collagen membrane, at desired time points (1, 3, 5, and 7 days), was incubated in CCK-8 solution in a 5% CO2 (v/v) incubator at 37 C for 3 h. The intense orange formazan derivative formed by cell metabolism was soluble in the culture medium. The absorbance was measured at 450 nm. Cell number was correlated with OD. 2.7. Cell migration in scaffolds The fibrin gel (Puji, Hangzhou, China) we used contains two parts. Fibrin solution A contains fibrinogen and sodium chloride solution, and fibrin solution B contains thrombin and calsium chloride solution. SDF-1 solution at 100 ng/mL was mixed with fibrin solution A (40 mg/mL) at a ratio of 1:2.5, and then mixed with fibrin solution B at a ratio of 1:1. The mixture with SDF-1 was then injected into the cylindrical cavity of the scaffold with a syringe. The fibrin gel formed in about 1 min. BMSCs were seeded on a 24-well non-adhesive plate at 2 105 cells/well, then the collagen cylinders with or without SDF-1 were placed on the plate and incubated at 37 C. After 24 h, the samples were washed 5 times with PBS and moved to a new 24well plate, then some samples were assessed using CCK-8. Others were cut into sections and cell nuclei were stained with hematoxylin-eosin (HE) or DAPI (Beyotime Institute of Biotechnology). Data were confirmed in 3 independent experiments.
2.9. Gross morphology and histology At 6 and 12 weeks after surgery, the rabbits were sacrificed by intravenous overdose of pentobarbital. Samples from each group were examined and photographed for evaluation according to the International Cartilage Repair Society (ICRS) macroscopic assessment scale for cartilage repair [32]. After gross examination, samples were fixed in 4% formalin, decalcified in 4% ethylenediamine tetraacetic acid for 14 days and then embedded in paraffin and cut into 7 mm sections. Sections from each sample were stained with Safranin O for glycosaminoglycan distribution. Histological and histomorphometric observation was performed under a light microscope (X71; Olympus, Tokyo, Japan) and analyzed with DP Controller 3.1.1.267 software (Olympus). Depth of the tissue protruding was scored, and all parameters were scored from 0 (not present) to 3 (abundantly present). For the overall evaluation of regenerated tissue in the defects, the repaired tissues were graded blindly by three observers, using the ICRS Visual Histological Assessment Scale [33].
2.10. Biomechanical evaluation Biomechanical evaluation was performed as below. Samples were placed in PBS at room temperature for 3e4 h to equilibrate before testing. The compressive mechanical properties of the surface cartilage layer were tested with an Instron testing machine (model 5543; Instron, Canton, MA) and software (Bluehill V2.0; Instron), using a 2 mm diameter cylindrical indenter fitted with a 10 N maximum loading cell. The unconfined equilibrium modulus was determined by applying a step displacement (20% strain) and monitoring compressive force with time until equilibrium was reached. The thickness of the fully relaxed cartilage layer was tested to estimate strain for applied deformations. The cross-head speed used was ~0.06 mm min1. The ratio of equilibrium force to cross-sectional area was divided by the applied strain to calculate the equilibrium modulus (in MPa). 16 samples following longterm treatment in vivo (12 weeks) were tested, and samples with sham operations were also evaluated.
2.11. Immunohistochemistry Paraffin sections (7 mm) were incubated with 0.5% pepsin (Sangon Biotech, Shanghai, China) in 5 mM HCl at 37 C for 30 min for antigen retrieval. Endogenous peroxidase was blocked by incubation with 3% hydrogen peroxide in methanol for 10 min. Nonspecific protein binding was blocked by incubation with 1% BSA. We primarily omitted the primary antibody and used 1% BSA as negative control. After overnight incubation at 4 C with primary antibodies (IgG, polyclonal) rabbit antiCol1 (Anbo, San Francisco, CA) and mouse anti-MMP13 (Millipore, Darmstadt, Germany), sections were incubated with goat anti-mouse (Beyotime Institute of Biotechnology Inc., Jiangsu, China) or goat anti-rabbit (Beyotime) secondary antibodies for 2 h at room temperature, then rinsed in distilled water (3 5 min) and incubated with DAB for 10 min at room temperature. The DAB substrate system (Zsbio, Beijing, China) was used for color development as the secondary antibodies have a peroxide group conjugated to DAB. Hematoxylin staining was used to reveal nuclei. The histomorphometry on immunohistochemistry was analyzed using the Image-Pro Plus software. The measurement parameters included sum of total area and integral optical density (IOD). The image was converted to gray scale image, and the values were quantified. The data was expressed as average IOD per area (IOD/ area) and normalized to the 6 weeks random group.
2.12. Statistical analysis All quantitative data are presented as mean ± SD. Multiple comparisons between the groups are analyzed using one-way ANOVA/Scheffe's post hoc test at 0.05 level. Student's t-test is used to assess the statistical significance of differences between two groups at significance level of 0.05.
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3. Results 3.1. Macroscopic and microscopic structure of the collagen scaffolds Two types of scaffolds with radially oriented and random pores were fabricated by thermal crosslinking and freeze-drying. Digital images showed that the scaffolds were sponge-like, and presented a bright white appearance (Fig. 1a). Our method succeeded in fabricating collagen scaffolds with radially oriented channels and random pores. Scanning electron microscopy of cross-sections
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showed radially oriented and aligned channels from the boundary to the central hole of the radially oriented scaffold. The scaffolds also consisted of oriented channels in longitudinal sections (Fig. 1b). Whereas in the random scaffold, disordered pores of 100e300 mm were uniformly distributed throughout the scaffold (Fig. 1c). 3.2. Mechanical properties, cytotoxicity tests and cell proliferation assays of scaffolds Since the compressive elastic modulus is not available for a cylindrical scaffold with radial channels in the radial direction, we use the value of compressive force/displacement (N/mm) instead to show change of the stress with different compression degrees. With a deformation range from 0% to 10%, the radially oriented scaffolds (0.143 ± 0.042 N/mm) had significantly better mechanical property compared with the random scaffolds (0.037 ± 0.010 N/ mm, Fig. 2a, P < 0.05). We tested the scaffolds with cytotoxicity tests (Fig. 2b) and cell proliferation assays (Fig. 2c). Rabbit BMSCs were seeded onto radially oriented and random scaffolds and cultured for 1 week. CCK-8 assays were carried out on days 1, 3, 5, and 7 after seeding. The cell viability results showed that the scaffolds supported cell growth well and were not toxic (Fig. 2b). Cell proliferation assay showed that BMSCs have a good proliferative capability on both radial and random scaffolds (Fig. 2c). The CCK-8 reading was slightly higher in the radially oriented group, but there was no significant difference between the radially oriented and random scaffolds. Collectively, our results suggested that the radially oriented collagen scaffold has better mechanical properties than the random scaffold, and both support cell proliferation well. 3.3. Radially oriented collagen scaffold with SDF-1 facilitates the migration of BMSCs To investigate the capacity of the scaffolds to promote cell migration, radially oriented or random scaffolds with or without SDF-1 were immersed in medium with suspensions of BMSCs in non-adhesive 24-well plates at 2 105 cells/well (Fig. 3a). After 24 h, the scaffolds were washed 5 times with PBS and processed for histology and CCK-8 analysis. We found the random scaffolds were compared more loosely with the radially oriented scaffolds by HE staining (Fig. 3b). Cell nuclei visualized by DAPI staining showed relatively more nuclei in radially oriented scaffolds (with and without SDF-1) than in random scaffolds (Fig. 3b,c). In addition, the largest number of cells was seen in the radially oriented þ SDF-1 group (Fig. 3b,c). The CCK-8 reading was also significantly higher in the radially oriented and radially oriented þ SDF-1 groups than in the random and random þ SDF-1 groups (Fig. 3d). These results indicated that the radially oriented collagen scaffold we fabricated promoted BMSC migration, and this effect was further facilitated by SDF-1. 3.4. Enhancement of osteochondral defect repair by the radially oriented collagen scaffold with SDF-1
Fig. 2. Mechanical properties, cytotoxicity tests and cell proliferation assays of scaffolds. (a) Mechanical property of the collagen scaffolds. (b) Cytotoxicity test of the collagen scaffolds. (c) Cell proliferation assay of the collagen scaffolds. n ¼ 3. RO, radially oriented scaffold. *p < 0.05; **p < 0.01; NS, not significant. (a, Student's t-test; b, analysis of variance (ANOVA)).
The in vitro data prompted us to further evaluate the effect of the radially oriented scaffold on cartilage regeneration in vivo. Random and radially oriented scaffolds with or without SDF-1 were transplanted into rabbit osteochondral defects. Six and twelve weeks post-surgery, neo-tissues were formed and the implanted scaffolds were not distinguishable from the newly-formed tissue under macroscopic observation (Fig. 5a). The adjacent cartilage from the edge of the defect showed tissue protruding in both the radial and random groups (Fig. 4c). However, protruding of the tissues was
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Fig. 3. Radially oriented collagen scaffold with SDF-1 facilitates the migration of BMSCs. (a) Model of BMSCs migration in scaffolds in vitro. (b) Hematoxylin-eosin and DAPI staining (yellow arrows indicate cell nuclei, photographed near the center of the scaffold). Scale bars, 200 um. (c) The cell number was quantified at low magnification. (d) CCK-8 assays of scaffolds with or without of SDF-1. n ¼ 3. SDF-1, stromal cell-derived factor-1; r þ SDF-1, random scaffold with SDF-1; RO þ SDF-1, radially oriented scaffold with SDF-1. *p < 0.05 (ANOVA). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
significantly deeper in radial scaffolds than in random scaffolds (Fig. 4d, P < 0.05). From the subchondral bone, we could find the neo-cartilage partly aligned with the direction of the radial scaffolds in the vertical direction (Fig. 4c). In random scaffolds neocartilage orientation was more disordered (Fig. 4c). These data partly indicated that the radially oriented collagen scaffold could facilitate the migration of cells from the edge of the defect and the growth of the cartilage from the subchondral bone.
As assessed macroscopically, the defects had become firm and smooth in radially oriented and radially oriented þ SDF-1 groups, while in random groups only a soft and friable tissue was presented (Fig. 5a). Macroscopic evaluation with International Cartilage Repair Society (ICRS) scoring showed substantial improvement with radially oriented scaffold implantation compared with the random scaffold at 6 and 12 weeks (p < 0.05) (Fig. 5b). ICRS scoring also showed that addition of SDF-1 greatly enhanced the efficacy of
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Fig. 4. Radially oriented collagen scaffold facilitates the migration of BMSCs and tissues protruding in vivo. (a) Experimental design for in vivo cartilage regeneration. Defects were filled with or without the scaffolds. (b) Model of the radially oriented scaffold in facilitating the migration of BMSCs and tissues protruding. (c) Safranin-O stain images of radially oriented and random group samples (6 weeks). Tissues protruding can be observed (indicated by the arrows). Edges and bottoms of the section were observed by higher magnification (indicated by asterisks). Neo-cartilage was partly aligned with the direction of the radial scaffolds in the vertical direction (indicated by the arrows). Scale bars, 500 mm. (d) Depth of the tissue protruding was scored. n ¼ 3. R, random group; R þ S, random scaffold with SDF-1 group; RO, radially oriented scaffold group; RO þ S, radially oriented scaffold with SDF-1 group. *p < 0.05 (ANOVA).
the radially oriented scaffold on cartilage regeneration at 6 weeks. Though the radially oriented þ SDF-1 group had a higher score, it was not significantly different from the radially oriented group at 12 weeks. The repaired tissue was then analyzed histologically. At 6 weeks after transplantation, there was fibrous tissue instead of cartilage formation in the joint surface of the defect in the random and random þ SDF-1 groups (Fig. 5c). In contrast, in the radially oriented group, the joint surface of the defect was repaired with a mixture of fibrous tissue and fibrocartilage as shown by Safranin-O staining. In the radially oriented þ SDF-1 group (Fig. 5c), the joint surface of the defect was smooth and as well was repaired with a mixture of fibrocartilage and cartilage-like tissue. The total histological score was 11.83 ± 0.62 in the radially oriented collagen group, which was markedly higher than the value of 0.50 ± 0.36 for the random collagen group (p < 0.05). Besides, the score was 3.70 ± 0.29 in the random þ SDF-1 group, higher than that of the random collagen group (p < 0.05). And the score was 16.33 ± 0.47 in the radially
oriented þ SDF-1 group, which was notably higher than the 11.83 ± 0.62 of in radially oriented collagen group (p < 0.05) (Fig. 5d). At 12 weeks after transplantation, the joint surfaces of the defect in the random and random þ SDF-1 groups were still filled with fibrous tissue (Fig. 5c). Notwithstanding, in the radially oriented and radially oriented þ SDF-1 groups (Fig. 5c), the joint surfaces of the defect were composed of thicker, hyaline cartilage-like tissue. Meanwhile, in the defect repaired by the radially oriented and radially oriented þ SDF-1 groups, we could find tissues protruding from the defect edges (as indicated by the lines) to the central defect area (as indicated by the arrows) (Fig. 5c). The total histological score was 17.00 ± 0.82 in the radially oriented collagen group, markedly higher than that of the random collagen group (2.60 ± 0.24; p < 0.05). And the score was 10.13 ± 0.66 in the random þ SDF-1 group, notably higher than that in the random collagen group (p < 0.05). However, there was no significant difference between the radially oriented collagen groups with and without SDF-1 (Fig. 5d).
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Fig. 5. Radially oriented collagen scaffold with SDF-1 for in vivo cartilage regeneration. (a) Gross findings with healed defects indicated by red arrows. (b) Macroscopic evaluation according to ICRS macroscopic scores. (c) Safranin-O staining images. The lines indicate the defect edges, and the arrows indicate the tissue protruding. Scale bars, 250 mm. (d) Evaluation according to the histological scoring system. (e) Young's moduli of the repaired tissues by mechanical test. n ¼ 3. r þ SDF-1, random scaffold with SDF-1 group; RO þ SDF1, radially oriented scaffold with SDF-1 group; CTL, control group. *p < 0.05 (ANOVA). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Together, these data indicated that cartilage regeneration with a combination of SDF-1 and the radially oriented scaffold is effective in repairing the cartilage defect in the rabbit model. 3.5. Biomechanical evaluation The Young's moduli of the repaired tissues from four groups at 12 weeks after surgery were determined and compared, and the tissues from rabbit knee joints with sham operations were used as controls. The compressive modulus of the repaired tissue in the radially oriented group (3.10 ± 0.16 MPa) showed better mechanical property than specimens from the random group (2.55 ± 0.58 MPa, P < 0.05), and the radially oriented þ SDF-1 group (4.06 ± 0.58 MPa) showed better mechanical property than the radially oriented group (3.57 ± 0.50 MPa, P < 0.05). Meanwhile, the radially oriented þ SDF-1 group had the highest compressive modulus, which was ~77% of the control group (Fig. 5e, Supplementary Fig. 1).
3.6. Effect of collagen scaffolds on expression of Col1 and MMP13 in vivo In addition to cartilage repair, we examined the effects of the radially oriented collagen scaffold combined with SDF-1 on expression of Col1 and MMP13 in tissue-engineered cartilage. The expression level of Col1 (a representative marker of ossification) was significantly higher in the random and random þ SDF-1 groups than the radially oriented and radially oriented þ SDF-1 groups at 6 and 12 weeks (Fig. 6a, P < 0.05). Moreover, compared with radially oriented group at 6 weeks, the radially oriented þ SDF-1 group significantly decreased the matrix Col1 levels (Fig. 6a, P < 0.05). These results indicated that the combination of radially oriented scaffold and SDF-1 protected repair tissues from ossification. In addition to inhibiting the expression of Col1, the radially oriented and radially oriented þ SDF-1 groups had significantly lower expression levels of MMP13 (a representative marker of cartilage
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Fig. 6. Immunohistochemical staining and quantitative data for ossification marker Col1 (a) and cartilage degradation marker MMP13 (b). Scale bars. 200 mm. n ¼ 3. *p < 0.05 (ANOVA).
degradation) at 6 and 12 weeks compared with the random and random þ SDF-1 groups (Fig. 6b, P < 0.05). Compared with radially oriented group at 6 weeks, the radially oriented þ SDF-1 group also significantly decreased the expression levels of MMP13 (Fig. 6b, P < 0.05). 4. Discussion In this study, we created a novel collagen scaffold with radially oriented channels and investigated its effect in combination with SDF-1 on facilitating cell homing and enhancing osteochondral repair in three steps. First, we characterized the radially oriented channel collagen scaffold; second, we showed that the radially oriented scaffold with SDF-1 modulated the migration of MSCs in vitro; and third, we tested the efficacy of the radially oriented channel collagen scaffold combined with SDF-1 on the healing of osteochondral defects in rabbits. Finally by the immunohistochemistry of ossification marker COL1 and cartilage degradation marker MMP13 [34,35], the radially oriented scaffold combined with SDF-1 could effectively inhibit expression of Col1 and MMP13 and therefore promote the formation of normally functional cartilage in tissue-engineered cartilage.
Several reports have shown that SDF-1 facilitates cell migration. Dar found that SDF-1 as well as its unique ligands is both expressed by BMSCs and therefore SDF-1 can regulate the migration of BMSCs [36]. Vagima discovered that in the course of stem cells homing to bone marrow, their retention, engraftment, and egress to the circulation involve SDF-1/CXCR4 interactions [37]. What is more, other stem-cell features such as survival and proliferation, also depend on the SDF-1/CXCR4 axis. In our experiments, the radially oriented scaffold with SDF-1 performed better in enhancing osteochondral repair than the random scaffold, as assessed by the aggregation of BMSCs and less expression of MMP13 and Col1. These results indicated that SDF-1 enhances BMSC migration. Moreover, SDF-1 might alter the features of BMSCs and hence benefit osteochondral repair. However, although adding SDF-1 can further improve the migration of BMSCs in vitro and the in vivo healing of cartilage defects, it is not the major contributing factor and less important compared with the orientation of scaffolds in this study (in vitro and in vivo). Few studies have been conducted to develop radially oriented biomaterials. Recently, a study using radial-like chitosan spheres showed that radially oriented material possesses good properties and can be used for biomedical applications [38]. In our experiments, both radially oriented and random structures were used
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as scaffolds to enhance osteochondral repair. Conforming to our expectations, the radially oriented structure exhibited a predetermined 3-D shape, as well as suitable porosity and micromechanical properties. Brouwer and colleagues designed a twolayered collagenous scaffold with a radial pore structure for repair of the diaphragm, and found that cells infiltrate further into the center of the scaffold [24,39]. This is consistent with our results that BMSCs were recruited into the radially oriented scaffold. Moreover, by direct or indirect interactions with SDF-1, BMSCs were better aggregated. Although the random structure possessed these features, the radially oriented structure had better mechanical property and promoted osteochondral defects healing better. Practical changes to scaffolds could be investigated for further use, as there are still some limitations of current scaffolds in this study. Further increases in cellular infiltration and tissue regeneration could be achieved not only by longer periods of implantation, but also by the addition of growth factors to the scaffolds [40]. Specific growth factors to attract and stimulate certain cell types may be attached to the scaffold, such as transforming growth factor-b and bone morphogenetic protein2, to increase proliferation and the production of cartilaginous extracellular matrix, or fibroblast growth factor-2 to increase proteoglycan production [4143]. Coating radially oriented scaffold structures with bio-adhesive components such as chondroitin sulfate could also increase their integrative efficiency [44]. Cartilage is a tissue which is difficult to regeneration. In this study, cartilage is regenerated by endogenous cells that are supplied from the defect areas. Hence we can believe that other tissues could also be regenerated without cell transplantation. These findings suggest a regenerative treatment for cartilage defects that can potentially be applied in patients with arthritis, trauma or osteonecrosis [8]. 5. Conclusions Radially oriented scaffolds have ordered and aligned channels in both the horizontal and the vertical direction. Hence, such scaffolds have better mechanical properties than random scaffolds. This material enhanced cell homing, and this effect was further facilitated by addition of SDF-1. Finally, in rabbits, the radially oriented scaffold in conjunction with SDF-1 was efficient in repairing a cartilage defect. Financial interests The authors hereby declare that no conflict of interest exists. Acknowledgments This work was supported by National key scientific research projects (2012CB966604), the National Natural Science Fund (81125014, 81101356, 81201395, 31000436), Zhejiang province public welfare fund (2012C3112), Zhejiang Provincial Natural Science Foundation of China (LY13C100001), Postdoctoral Foundation of China (2013M531465), as well as sponsored by Regenerative Medicine in Innovative Medical Subjects of Zhejiang Province and Zhejiang Provincial Program for the Cultivation of High-level Innovative Health talents. The authors thank Mr. Hanmin Chen for SEM imaging, the Imaging Center of Zhejiang University School of Medicine for technical assistance and Mrs. Shufang Zhang for technical assistance.
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P. Chen et al. / Biomaterials 39 (2015) 114e123 [26] Theiss HD, Vallaster M, Rischpler C, Krieg L, Zaruba MM, Brunner S, et al. Dual stem cell therapy after myocardial infarction acts specifically by enhanced homing via the SDF-1/CXCR4 axis. Stem Cell Res 2011;7(3):244e55. [27] Shichinohe H, Kuroda S, Yano S, Hida K, Iwasaki Y. Role of SDF-1/CXCR4 system in survival and migration of bone marrow stromal cells after transplantation into mice cerebral infarct. Brain Res 2007;1183:138e47. [28] Lau TT, Wang DA. Stromal cell-derived factor-1 (SDF-1): homing factor for engineered regenerative medicine. Expert Opin Biol Ther 2011;11(2):189e97. [29] MacArthur Jr JW, Purcell BP, Shudo Y, Cohen JE, Fairman A, Trubelja A, et al. Sustained release of engineered stromal cell-derived factor 1-alpha from injectable hydrogels effectively recruits endothelial progenitor cells and preserves ventricular function after myocardial infarction. Circulation 2013;128(11 Suppl. 1):S79e86. [30] Shen W, Chen X, Chen J, Yin Z, Heng BC, Chen W, et al. The effect of incorporation of exogenous stromal cell-derived factor-1 alpha within a knitted silk-collagen sponge scaffold on tendon regeneration. Biomaterials 2010;31(28):7239e49. [31] Ouyang HW, Goh JC, Thambyah A, Teoh SH, Lee EH. Knitted poly-lactide-coglycolide scaffold loaded with bone marrow stromal cells in repair and regeneration of rabbit Achilles tendon. Tissue Eng 2003;9(3):431e9. [32] van den Borne MP, Raijmakers NJ, Vanlauwe J, Victor J, de Jong SN, Bellemans J, et al. International Cartilage Repair Society (ICRS) and Oswestry macroscopic cartilage evaluation scores validated for use in autologous chondrocyte implantation (ACI) and microfracture. OARS Osteoarthr Res Soc 2007;15(12):1397e402. [33] Mainil-Varlet P, Aigner T, Brittberg M, Bullough P, Hollander A, Hunziker E, et al. Histological assessment of cartilage repair: a report by the Histology Endpoint Committee of the International Cartilage Repair Society (ICRS). J Bone Jt Surg Am 2003;(85-A Suppl. 2):45e57. [34] Han M, Yang X, Lee J, Allan CH, Muneoka K. Development and regeneration of the neonatal digit tip in mice. Dev Biol N Y 1985 2008;315(1):125e35. [35] Piecha D, Weik J, Kheil H, Becher G, Timmermann A, Jaworski A, et al. Novel selective MMP-13 inhibitors reduce collagen degradation in bovine articular and human osteoarthritis cartilage explants. Inflamm Res 2010;59(5):379e89.
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Radially oriented collagen scaffold with SDF-1 promotes osteochondral repair by facilitating cell homing Pengfei Chen a, b, 1, Jiadong Tao a, 1, Shouan Zhu a, 1, Youzhi Cai a, Qijiang Mao a, Dongsheng Yu a, Jun Dai a, HongWei Ouyang a, * a
Center for Stem Cell and Tissue Engineering, School of Medicine, Zhejiang Provincial Key Lab for Tissue Engineering and Regenerative Medicine, Zhejiang University, 866 Yu Hang Tang Road, Hangzhou 310058, China Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, 3 East Qingchun Road, Hangzhou 310016, China
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 July 2014 Accepted 19 October 2014 Available online
The migration of cells from the side and the bottom of the defect is important in osteochondral defect healing. Here, we designed a novel collagen scaffold that possessed channels in both the horizontal and the vertical directions, along with stromal cell-derived factor-1 (SDF-1) to enhance osteochondral regeneration by facilitating cell homing. Firstly we fabricated the radially oriented and random collagen scaffolds, then tested their properties. The radially oriented collagen scaffold had better mechanical properties than the random scaffold, but both supported cell proliferation well. Then we measured the migration of BMSCs in the scaffolds in vitro. The radially oriented collagen scaffold effectively promoted their migration, and this effect was further facilitated by addition of SDF-1. Moreover, we created osteochondral defects in rabbits, and implanted them with random or oriented collagen scaffolds with or without SDF-1, and evaluated cartilage and subchondral bone regeneration at 6 and 12 weeks after surgery. Cartilage regeneration with both the radially oriented scaffold and SDF-1 effectively promoted repair of the cartilage defect. Our results confirmed that the implantation of the radially oriented channel collagen scaffold with SDF-1 could be a promising strategy for osteochondral repair. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Osteochondral tissue engineering Cartilage defect repair SDF-1 Collagen scaffolds
1. Introduction Tissue or organ defect is the final clinical manifestation of all refractory diseases. For decades, cell-based methods have been used to substitute pathologic cells and to cure tissue defects [15]. However, cell delivery has shortcomings such as immune rejection, pathogen transmission, potential tumorigenesis, issues with packaging, storage, and difficulties in clinical adoption and regulatory approval [68]. Osteoarthritis (OA), the most common form of arthritis, is a group of mechanical abnormalities resulting in the articular cartilage degradation [9]. Though cell transplantation for cartilage regeneration is well studied [1013], defect regeneration by cell homing, especially without cell transplantation, remains a promising approach lacking experimental testing [1416].
* Corresponding author. Mailbox #39, Center for Stem Cell and Tissue Engineering, School of Medicine, Zhejiang University, #866 Yu Hang Tang Road, Hangzhou 310058, China. Tel./fax: þ86 571 88208262. E-mail address: [email protected] (H. Ouyang). 1 Authors contribute equally to this work. http://dx.doi.org/10.1016/j.biomaterials.2014.10.049 0142-9612/© 2014 Elsevier Ltd. All rights reserved.
The predominant model of cartilage regeneration is focal lesions such as osteochondral defects [17]. It is generally accepted that osteochondral defect healing is developed from the bottom of the defect where bone marrow-derived MSCs (BMSCs) contribute to the regeneration. However, previous serial cartilage studies suggest that during cartilage repair segmental neo-cartilage is formed by adjacent tissue protruding [1820]. These findings suggest that the migration of cells from the sides of defect is also important in osteochondral defect healing. Thus the choices of an appropriate scaffold which can facilitate cell migration are of great importance. The utilization of biphasic and multiphasic scaffolds to replicate the multiphasic nature of the native tissue is the core challenge and precondition for the organization of living cells to functional tissue. Designing scaffolds with tissue-specific architecture is an effective way for osteochondral repair [2123]. Meanwhile the threedimensional environment of the scaffolds such as pore size and orientation will affect the extracellular matrix deposition, as cells infiltrate into the scaffold will align with the pore and follow the channels arranged by the scaffold [24]. So it seems that current popular osteochondral scaffolds with random structures cannot facilitate the process of cell homing. There is, therefore, a critical
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need to modify the structural properties of scaffolds in a radially oriented manner to achieve satisfactory outcomes in cartilage repair. Here, we have designed a novel radially oriented collagen scaffold, which has oriented channels in both the horizontal and the vertical directions. We further assessed the therapeutic effects of this biological material for osteochondral repair. SDF-1, the unique ligand of its specific receptor, chemokine (CX-C motif) receptor 4 (CXCR-4), is constitutively expressed in many organs [25]. Studies have demonstrated that SDF-1 stimulates stem-cell homing [26] and migration [25]. Bone marrow stromal cells (BMSCs), express both SDF-1 and CXCR-4 and, hence, BMSCs promote their own survival and proliferation through an autocrine or paracrine mechanism [27]. The general approach for SDF-1 delivery in tissue engineering is direct protein incorporation into scaffolds which has succeeded in improving the regeneration of tissue or organ defect [28,29]. Our previous studies also demonstrated that SDF-1 is one of the key factors for MSC migration in the subchondral bone environment and can be used osteochondral repair [19,30]. In this study, we hypothesized that our radially oriented collagen scaffold, along with SDF-1, promoted osteochondral repair by facilitating cell homing. To test this hypothesis, experiments
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were designed as follows: (1) fabrication and characterization of a radially oriented channel collagen scaffold; (2) in vitro testing of the efficiency of this scaffold along with SDF-1 in promoting MSC migration; and (3) in vivo analysis of the effect of this scaffold with SDF-1 on the healing of osteochondral defects in rabbits. 2. Materials and methods 2.1. Materials Jinhua pigs (<100 kg, <6-month-old) of either gender were used as donors. Type I collagen was isolated and purified from pig tendon in our lab. Reagents were from Sigma unless specifically indicated. 2.2. Fabrication of the radially oriented and random collagen scaffold The radially oriented scaffold was made using a temperature gradient-guided thermal-induced phase-separation technique (Fig. 1a). The pig tendons were dissected out, sliced and washed with several changes of cold distilled water to remove plasma proteins, and then extracted with 1 M NaCl in 50 mM TriseHCl (pH 7.4) and constantly stirred overnight at 4 C. The supernatant was decanted and the remainder was washed with several changes of cold distilled water to remove salts. Then the tendon was incubated in 0.5 M acetic acid (1.0% w/v) with 1 mg/ml pepsin overnight at 4 C to obtain an aqueous extract. The extract was precipitated with saline (0.9 M NaCl) and dissolved in 0.5 M HAc. The solution was then dialyzed, frozen and freeze-dried to obtain the collagen. The collagen was dissolved again in 0.05 M acetic acid to form 10 mg/ml solution. Then the collagen solution was added to a
Fig. 1. Macroscopic and microscopic structures of the collagen scaffolds. (a) Fabrication of the radially oriented and random collagen scaffolds. (b) Microscopic structure of the collagen scaffolds.
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mold with an inner diameter of 5.0 mm and height of 20.0 mm. The wall of the mold was made of copper, which was at a temperature of 20 C as a cooling source. In the middle of the mold, there was a plastic rod 1.5 mm in diameter. The top and bottom of the mold was also made of plastic. Both the top and bottom were designed as thermal insulation material. With evaporation of the liquid nitrogen, the collagen solution was unidirectionally frozen from the edge to the center of the solution. The radially oriented collagen crystallization occurred in half an hour. For the random scaffold, the collagen solution was added into a mold of the same size. But the top and bottom were made of copper. After addition of the collagen solution, the mold was put into liquid nitrogen at 196 C for 10 min. Then random collagen crystallization occurred. Next, both the radially oriented and randomly crystallized collagen were kept at 80 C for >4 h. The crystallized materials were removed from the molds and lyophilized in a freeze-dryer under vacuum for 48 h. Then the scaffolds were cut into cylinders 5.0 mm high and subjected to thermal crosslinking, then exposed to 60Co radiation for sterilization before cell culture and in vivo experimentation. Briefly, the thermal crosslinking was carried out at 25 C for 24 h, 110 C for 72 h, and 65 C for 48 h.
2.8. Animal model All animals were treated according to the standard guidelines approved by Zhejiang University Ethics Committee (ZJU 2010105003). 36 adult male New Zealand white rabbits (2.5e3 kg) were maintained singly in stainless-steel cages. After intramuscular and intravenous injection with 10% chloral hydrate (4 ml kg1), the knee joint was opened using a medial parapatellar approach. The patella was dislocated laterally and the surface of the femoropatellar groove exposed. A cylindrical osteochondral defect 4 mm in diameter and 3.5e4 mm deep was created in the patellar groove using a stainless-steel punch. The defect in the left joint was treated with a random scaffold or a radially oriented scaffold, and the right joint was treated with a random scaffold with SDF-1 or a radially oriented scaffold with SDF-1 (n ¼ 14 joints in each group). 8 rabbits were treated with sham operations as controls. The joint capsule and skin were closed with interrupted sutures. Immediately after surgery, the animals were returned to their individual cages without joint immobilization. A postoperative antibiotic (gentamicin) was administered intramuscularly at 400,000 U per day for 3 days. After sacrifice at 6 and 12 weeks, knee joints from each group were evaluated histologically.
2.3. Characterization of the radially oriented and random collagen scaffolds The collagen scaffold samples were cut in half in both vertical and cross-section, mounted on aluminum stubs, sputter-coated with gold, and observed under a scanning electron microscope (Hitachi S3000N) at an accelerating voltage of 15 kV. The mechanical properties of the scaffolds were tested using an Instron testing machine (model 5543; Instron, Canton, MA) and software (Bluehill V2.0; Instron). It was a compression testing at a speed of 2.0 mm/min (without the tare load). According to the normal range of deformation, the deformation range of 0%e10% was chosen. To show the change of the stress with different compression degrees, the scaffold with a height of 5.0 ± 0.2 mm was placed horizontally between two mechanical detecting heads during the testing. 12 samples from the random and radially oriented scaffolds were tested. 2.4. Isolation and culture of bone marrow mesenchymal stem cells BMSCs were isolated by short-term adherence to plastic as described previously [31]. Rabbit bone marrow was aspirated from the iliac crest of 4-month-old female New Zealand white rabbits. Nucleated cells were isolated by centrifugation at 1200 rpm for 5 min and the supernatant was removed. Following this, the nucleated cell layer was carefully collected and re-suspended in culture medium containing Dulbecco's modified Eagle's medium (DMEM), 10% w/v fetal bovine serum (FBS), 100 units/ml penicillin, and 100 mg/ml streptomycin (all from Gibco). Cells were plated at 5 106 per 100 mm dish and incubated at 37 C under 5% humidified CO2. After 48 h, non-adherent cells were discarded and adherent cells were continuously cultured. The medium was changed every 3 days. When cells became nearly confluent, they were detached with trypsin and serially sub-cultured. 2.5. Cell viability assay A 0.4 cm diameter collagen membrane was immersed in 0.12 ml DMEM-high glucose containing 10% FBS and 1% penicillin/streptomycin for >24 h. BMSCs were seeded on a 96-well plate at 103 cells/well. After 24 h, the medium of the test group was replaced with leaching liquor acquired from collagen membrane or fresh DMEM-high glucose containing 10% FBS and 1% penicillin/streptomycin or 5% DMSO. After 3 days, the cytotoxicity of the collagen membrane was assessed using Cell Counting KIT-8 (CCK-8, Dojindo, Kumamoto, Japan) at 450 nm. Cell number was correlated with optical density (OD). 2.6. Cell proliferation assay Cell proliferation was measured using CCK-8. The BMSC-seeded collagen membrane, at desired time points (1, 3, 5, and 7 days), was incubated in CCK-8 solution in a 5% CO2 (v/v) incubator at 37 C for 3 h. The intense orange formazan derivative formed by cell metabolism was soluble in the culture medium. The absorbance was measured at 450 nm. Cell number was correlated with OD. 2.7. Cell migration in scaffolds The fibrin gel (Puji, Hangzhou, China) we used contains two parts. Fibrin solution A contains fibrinogen and sodium chloride solution, and fibrin solution B contains thrombin and calsium chloride solution. SDF-1 solution at 100 ng/mL was mixed with fibrin solution A (40 mg/mL) at a ratio of 1:2.5, and then mixed with fibrin solution B at a ratio of 1:1. The mixture with SDF-1 was then injected into the cylindrical cavity of the scaffold with a syringe. The fibrin gel formed in about 1 min. BMSCs were seeded on a 24-well non-adhesive plate at 2 105 cells/well, then the collagen cylinders with or without SDF-1 were placed on the plate and incubated at 37 C. After 24 h, the samples were washed 5 times with PBS and moved to a new 24well plate, then some samples were assessed using CCK-8. Others were cut into sections and cell nuclei were stained with hematoxylin-eosin (HE) or DAPI (Beyotime Institute of Biotechnology). Data were confirmed in 3 independent experiments.
2.9. Gross morphology and histology At 6 and 12 weeks after surgery, the rabbits were sacrificed by intravenous overdose of pentobarbital. Samples from each group were examined and photographed for evaluation according to the International Cartilage Repair Society (ICRS) macroscopic assessment scale for cartilage repair [32]. After gross examination, samples were fixed in 4% formalin, decalcified in 4% ethylenediamine tetraacetic acid for 14 days and then embedded in paraffin and cut into 7 mm sections. Sections from each sample were stained with Safranin O for glycosaminoglycan distribution. Histological and histomorphometric observation was performed under a light microscope (X71; Olympus, Tokyo, Japan) and analyzed with DP Controller 3.1.1.267 software (Olympus). Depth of the tissue protruding was scored, and all parameters were scored from 0 (not present) to 3 (abundantly present). For the overall evaluation of regenerated tissue in the defects, the repaired tissues were graded blindly by three observers, using the ICRS Visual Histological Assessment Scale [33].
2.10. Biomechanical evaluation Biomechanical evaluation was performed as below. Samples were placed in PBS at room temperature for 3e4 h to equilibrate before testing. The compressive mechanical properties of the surface cartilage layer were tested with an Instron testing machine (model 5543; Instron, Canton, MA) and software (Bluehill V2.0; Instron), using a 2 mm diameter cylindrical indenter fitted with a 10 N maximum loading cell. The unconfined equilibrium modulus was determined by applying a step displacement (20% strain) and monitoring compressive force with time until equilibrium was reached. The thickness of the fully relaxed cartilage layer was tested to estimate strain for applied deformations. The cross-head speed used was ~0.06 mm min1. The ratio of equilibrium force to cross-sectional area was divided by the applied strain to calculate the equilibrium modulus (in MPa). 16 samples following longterm treatment in vivo (12 weeks) were tested, and samples with sham operations were also evaluated.
2.11. Immunohistochemistry Paraffin sections (7 mm) were incubated with 0.5% pepsin (Sangon Biotech, Shanghai, China) in 5 mM HCl at 37 C for 30 min for antigen retrieval. Endogenous peroxidase was blocked by incubation with 3% hydrogen peroxide in methanol for 10 min. Nonspecific protein binding was blocked by incubation with 1% BSA. We primarily omitted the primary antibody and used 1% BSA as negative control. After overnight incubation at 4 C with primary antibodies (IgG, polyclonal) rabbit antiCol1 (Anbo, San Francisco, CA) and mouse anti-MMP13 (Millipore, Darmstadt, Germany), sections were incubated with goat anti-mouse (Beyotime Institute of Biotechnology Inc., Jiangsu, China) or goat anti-rabbit (Beyotime) secondary antibodies for 2 h at room temperature, then rinsed in distilled water (3 5 min) and incubated with DAB for 10 min at room temperature. The DAB substrate system (Zsbio, Beijing, China) was used for color development as the secondary antibodies have a peroxide group conjugated to DAB. Hematoxylin staining was used to reveal nuclei. The histomorphometry on immunohistochemistry was analyzed using the Image-Pro Plus software. The measurement parameters included sum of total area and integral optical density (IOD). The image was converted to gray scale image, and the values were quantified. The data was expressed as average IOD per area (IOD/ area) and normalized to the 6 weeks random group.
2.12. Statistical analysis All quantitative data are presented as mean ± SD. Multiple comparisons between the groups are analyzed using one-way ANOVA/Scheffe's post hoc test at 0.05 level. Student's t-test is used to assess the statistical significance of differences between two groups at significance level of 0.05.
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3. Results 3.1. Macroscopic and microscopic structure of the collagen scaffolds Two types of scaffolds with radially oriented and random pores were fabricated by thermal crosslinking and freeze-drying. Digital images showed that the scaffolds were sponge-like, and presented a bright white appearance (Fig. 1a). Our method succeeded in fabricating collagen scaffolds with radially oriented channels and random pores. Scanning electron microscopy of cross-sections
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showed radially oriented and aligned channels from the boundary to the central hole of the radially oriented scaffold. The scaffolds also consisted of oriented channels in longitudinal sections (Fig. 1b). Whereas in the random scaffold, disordered pores of 100e300 mm were uniformly distributed throughout the scaffold (Fig. 1c). 3.2. Mechanical properties, cytotoxicity tests and cell proliferation assays of scaffolds Since the compressive elastic modulus is not available for a cylindrical scaffold with radial channels in the radial direction, we use the value of compressive force/displacement (N/mm) instead to show change of the stress with different compression degrees. With a deformation range from 0% to 10%, the radially oriented scaffolds (0.143 ± 0.042 N/mm) had significantly better mechanical property compared with the random scaffolds (0.037 ± 0.010 N/ mm, Fig. 2a, P < 0.05). We tested the scaffolds with cytotoxicity tests (Fig. 2b) and cell proliferation assays (Fig. 2c). Rabbit BMSCs were seeded onto radially oriented and random scaffolds and cultured for 1 week. CCK-8 assays were carried out on days 1, 3, 5, and 7 after seeding. The cell viability results showed that the scaffolds supported cell growth well and were not toxic (Fig. 2b). Cell proliferation assay showed that BMSCs have a good proliferative capability on both radial and random scaffolds (Fig. 2c). The CCK-8 reading was slightly higher in the radially oriented group, but there was no significant difference between the radially oriented and random scaffolds. Collectively, our results suggested that the radially oriented collagen scaffold has better mechanical properties than the random scaffold, and both support cell proliferation well. 3.3. Radially oriented collagen scaffold with SDF-1 facilitates the migration of BMSCs To investigate the capacity of the scaffolds to promote cell migration, radially oriented or random scaffolds with or without SDF-1 were immersed in medium with suspensions of BMSCs in non-adhesive 24-well plates at 2 105 cells/well (Fig. 3a). After 24 h, the scaffolds were washed 5 times with PBS and processed for histology and CCK-8 analysis. We found the random scaffolds were compared more loosely with the radially oriented scaffolds by HE staining (Fig. 3b). Cell nuclei visualized by DAPI staining showed relatively more nuclei in radially oriented scaffolds (with and without SDF-1) than in random scaffolds (Fig. 3b,c). In addition, the largest number of cells was seen in the radially oriented þ SDF-1 group (Fig. 3b,c). The CCK-8 reading was also significantly higher in the radially oriented and radially oriented þ SDF-1 groups than in the random and random þ SDF-1 groups (Fig. 3d). These results indicated that the radially oriented collagen scaffold we fabricated promoted BMSC migration, and this effect was further facilitated by SDF-1. 3.4. Enhancement of osteochondral defect repair by the radially oriented collagen scaffold with SDF-1
Fig. 2. Mechanical properties, cytotoxicity tests and cell proliferation assays of scaffolds. (a) Mechanical property of the collagen scaffolds. (b) Cytotoxicity test of the collagen scaffolds. (c) Cell proliferation assay of the collagen scaffolds. n ¼ 3. RO, radially oriented scaffold. *p < 0.05; **p < 0.01; NS, not significant. (a, Student's t-test; b, analysis of variance (ANOVA)).
The in vitro data prompted us to further evaluate the effect of the radially oriented scaffold on cartilage regeneration in vivo. Random and radially oriented scaffolds with or without SDF-1 were transplanted into rabbit osteochondral defects. Six and twelve weeks post-surgery, neo-tissues were formed and the implanted scaffolds were not distinguishable from the newly-formed tissue under macroscopic observation (Fig. 5a). The adjacent cartilage from the edge of the defect showed tissue protruding in both the radial and random groups (Fig. 4c). However, protruding of the tissues was
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Fig. 3. Radially oriented collagen scaffold with SDF-1 facilitates the migration of BMSCs. (a) Model of BMSCs migration in scaffolds in vitro. (b) Hematoxylin-eosin and DAPI staining (yellow arrows indicate cell nuclei, photographed near the center of the scaffold). Scale bars, 200 um. (c) The cell number was quantified at low magnification. (d) CCK-8 assays of scaffolds with or without of SDF-1. n ¼ 3. SDF-1, stromal cell-derived factor-1; r þ SDF-1, random scaffold with SDF-1; RO þ SDF-1, radially oriented scaffold with SDF-1. *p < 0.05 (ANOVA). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
significantly deeper in radial scaffolds than in random scaffolds (Fig. 4d, P < 0.05). From the subchondral bone, we could find the neo-cartilage partly aligned with the direction of the radial scaffolds in the vertical direction (Fig. 4c). In random scaffolds neocartilage orientation was more disordered (Fig. 4c). These data partly indicated that the radially oriented collagen scaffold could facilitate the migration of cells from the edge of the defect and the growth of the cartilage from the subchondral bone.
As assessed macroscopically, the defects had become firm and smooth in radially oriented and radially oriented þ SDF-1 groups, while in random groups only a soft and friable tissue was presented (Fig. 5a). Macroscopic evaluation with International Cartilage Repair Society (ICRS) scoring showed substantial improvement with radially oriented scaffold implantation compared with the random scaffold at 6 and 12 weeks (p < 0.05) (Fig. 5b). ICRS scoring also showed that addition of SDF-1 greatly enhanced the efficacy of
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Fig. 4. Radially oriented collagen scaffold facilitates the migration of BMSCs and tissues protruding in vivo. (a) Experimental design for in vivo cartilage regeneration. Defects were filled with or without the scaffolds. (b) Model of the radially oriented scaffold in facilitating the migration of BMSCs and tissues protruding. (c) Safranin-O stain images of radially oriented and random group samples (6 weeks). Tissues protruding can be observed (indicated by the arrows). Edges and bottoms of the section were observed by higher magnification (indicated by asterisks). Neo-cartilage was partly aligned with the direction of the radial scaffolds in the vertical direction (indicated by the arrows). Scale bars, 500 mm. (d) Depth of the tissue protruding was scored. n ¼ 3. R, random group; R þ S, random scaffold with SDF-1 group; RO, radially oriented scaffold group; RO þ S, radially oriented scaffold with SDF-1 group. *p < 0.05 (ANOVA).
the radially oriented scaffold on cartilage regeneration at 6 weeks. Though the radially oriented þ SDF-1 group had a higher score, it was not significantly different from the radially oriented group at 12 weeks. The repaired tissue was then analyzed histologically. At 6 weeks after transplantation, there was fibrous tissue instead of cartilage formation in the joint surface of the defect in the random and random þ SDF-1 groups (Fig. 5c). In contrast, in the radially oriented group, the joint surface of the defect was repaired with a mixture of fibrous tissue and fibrocartilage as shown by Safranin-O staining. In the radially oriented þ SDF-1 group (Fig. 5c), the joint surface of the defect was smooth and as well was repaired with a mixture of fibrocartilage and cartilage-like tissue. The total histological score was 11.83 ± 0.62 in the radially oriented collagen group, which was markedly higher than the value of 0.50 ± 0.36 for the random collagen group (p < 0.05). Besides, the score was 3.70 ± 0.29 in the random þ SDF-1 group, higher than that of the random collagen group (p < 0.05). And the score was 16.33 ± 0.47 in the radially
oriented þ SDF-1 group, which was notably higher than the 11.83 ± 0.62 of in radially oriented collagen group (p < 0.05) (Fig. 5d). At 12 weeks after transplantation, the joint surfaces of the defect in the random and random þ SDF-1 groups were still filled with fibrous tissue (Fig. 5c). Notwithstanding, in the radially oriented and radially oriented þ SDF-1 groups (Fig. 5c), the joint surfaces of the defect were composed of thicker, hyaline cartilage-like tissue. Meanwhile, in the defect repaired by the radially oriented and radially oriented þ SDF-1 groups, we could find tissues protruding from the defect edges (as indicated by the lines) to the central defect area (as indicated by the arrows) (Fig. 5c). The total histological score was 17.00 ± 0.82 in the radially oriented collagen group, markedly higher than that of the random collagen group (2.60 ± 0.24; p < 0.05). And the score was 10.13 ± 0.66 in the random þ SDF-1 group, notably higher than that in the random collagen group (p < 0.05). However, there was no significant difference between the radially oriented collagen groups with and without SDF-1 (Fig. 5d).
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Fig. 5. Radially oriented collagen scaffold with SDF-1 for in vivo cartilage regeneration. (a) Gross findings with healed defects indicated by red arrows. (b) Macroscopic evaluation according to ICRS macroscopic scores. (c) Safranin-O staining images. The lines indicate the defect edges, and the arrows indicate the tissue protruding. Scale bars, 250 mm. (d) Evaluation according to the histological scoring system. (e) Young's moduli of the repaired tissues by mechanical test. n ¼ 3. r þ SDF-1, random scaffold with SDF-1 group; RO þ SDF1, radially oriented scaffold with SDF-1 group; CTL, control group. *p < 0.05 (ANOVA). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Together, these data indicated that cartilage regeneration with a combination of SDF-1 and the radially oriented scaffold is effective in repairing the cartilage defect in the rabbit model. 3.5. Biomechanical evaluation The Young's moduli of the repaired tissues from four groups at 12 weeks after surgery were determined and compared, and the tissues from rabbit knee joints with sham operations were used as controls. The compressive modulus of the repaired tissue in the radially oriented group (3.10 ± 0.16 MPa) showed better mechanical property than specimens from the random group (2.55 ± 0.58 MPa, P < 0.05), and the radially oriented þ SDF-1 group (4.06 ± 0.58 MPa) showed better mechanical property than the radially oriented group (3.57 ± 0.50 MPa, P < 0.05). Meanwhile, the radially oriented þ SDF-1 group had the highest compressive modulus, which was ~77% of the control group (Fig. 5e, Supplementary Fig. 1).
3.6. Effect of collagen scaffolds on expression of Col1 and MMP13 in vivo In addition to cartilage repair, we examined the effects of the radially oriented collagen scaffold combined with SDF-1 on expression of Col1 and MMP13 in tissue-engineered cartilage. The expression level of Col1 (a representative marker of ossification) was significantly higher in the random and random þ SDF-1 groups than the radially oriented and radially oriented þ SDF-1 groups at 6 and 12 weeks (Fig. 6a, P < 0.05). Moreover, compared with radially oriented group at 6 weeks, the radially oriented þ SDF-1 group significantly decreased the matrix Col1 levels (Fig. 6a, P < 0.05). These results indicated that the combination of radially oriented scaffold and SDF-1 protected repair tissues from ossification. In addition to inhibiting the expression of Col1, the radially oriented and radially oriented þ SDF-1 groups had significantly lower expression levels of MMP13 (a representative marker of cartilage
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Fig. 6. Immunohistochemical staining and quantitative data for ossification marker Col1 (a) and cartilage degradation marker MMP13 (b). Scale bars. 200 mm. n ¼ 3. *p < 0.05 (ANOVA).
degradation) at 6 and 12 weeks compared with the random and random þ SDF-1 groups (Fig. 6b, P < 0.05). Compared with radially oriented group at 6 weeks, the radially oriented þ SDF-1 group also significantly decreased the expression levels of MMP13 (Fig. 6b, P < 0.05). 4. Discussion In this study, we created a novel collagen scaffold with radially oriented channels and investigated its effect in combination with SDF-1 on facilitating cell homing and enhancing osteochondral repair in three steps. First, we characterized the radially oriented channel collagen scaffold; second, we showed that the radially oriented scaffold with SDF-1 modulated the migration of MSCs in vitro; and third, we tested the efficacy of the radially oriented channel collagen scaffold combined with SDF-1 on the healing of osteochondral defects in rabbits. Finally by the immunohistochemistry of ossification marker COL1 and cartilage degradation marker MMP13 [34,35], the radially oriented scaffold combined with SDF-1 could effectively inhibit expression of Col1 and MMP13 and therefore promote the formation of normally functional cartilage in tissue-engineered cartilage.
Several reports have shown that SDF-1 facilitates cell migration. Dar found that SDF-1 as well as its unique ligands is both expressed by BMSCs and therefore SDF-1 can regulate the migration of BMSCs [36]. Vagima discovered that in the course of stem cells homing to bone marrow, their retention, engraftment, and egress to the circulation involve SDF-1/CXCR4 interactions [37]. What is more, other stem-cell features such as survival and proliferation, also depend on the SDF-1/CXCR4 axis. In our experiments, the radially oriented scaffold with SDF-1 performed better in enhancing osteochondral repair than the random scaffold, as assessed by the aggregation of BMSCs and less expression of MMP13 and Col1. These results indicated that SDF-1 enhances BMSC migration. Moreover, SDF-1 might alter the features of BMSCs and hence benefit osteochondral repair. However, although adding SDF-1 can further improve the migration of BMSCs in vitro and the in vivo healing of cartilage defects, it is not the major contributing factor and less important compared with the orientation of scaffolds in this study (in vitro and in vivo). Few studies have been conducted to develop radially oriented biomaterials. Recently, a study using radial-like chitosan spheres showed that radially oriented material possesses good properties and can be used for biomedical applications [38]. In our experiments, both radially oriented and random structures were used
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as scaffolds to enhance osteochondral repair. Conforming to our expectations, the radially oriented structure exhibited a predetermined 3-D shape, as well as suitable porosity and micromechanical properties. Brouwer and colleagues designed a twolayered collagenous scaffold with a radial pore structure for repair of the diaphragm, and found that cells infiltrate further into the center of the scaffold [24,39]. This is consistent with our results that BMSCs were recruited into the radially oriented scaffold. Moreover, by direct or indirect interactions with SDF-1, BMSCs were better aggregated. Although the random structure possessed these features, the radially oriented structure had better mechanical property and promoted osteochondral defects healing better. Practical changes to scaffolds could be investigated for further use, as there are still some limitations of current scaffolds in this study. Further increases in cellular infiltration and tissue regeneration could be achieved not only by longer periods of implantation, but also by the addition of growth factors to the scaffolds [40]. Specific growth factors to attract and stimulate certain cell types may be attached to the scaffold, such as transforming growth factor-b and bone morphogenetic protein2, to increase proliferation and the production of cartilaginous extracellular matrix, or fibroblast growth factor-2 to increase proteoglycan production [4143]. Coating radially oriented scaffold structures with bio-adhesive components such as chondroitin sulfate could also increase their integrative efficiency [44]. Cartilage is a tissue which is difficult to regeneration. In this study, cartilage is regenerated by endogenous cells that are supplied from the defect areas. Hence we can believe that other tissues could also be regenerated without cell transplantation. These findings suggest a regenerative treatment for cartilage defects that can potentially be applied in patients with arthritis, trauma or osteonecrosis [8]. 5. Conclusions Radially oriented scaffolds have ordered and aligned channels in both the horizontal and the vertical direction. Hence, such scaffolds have better mechanical properties than random scaffolds. This material enhanced cell homing, and this effect was further facilitated by addition of SDF-1. Finally, in rabbits, the radially oriented scaffold in conjunction with SDF-1 was efficient in repairing a cartilage defect. Financial interests The authors hereby declare that no conflict of interest exists. Acknowledgments This work was supported by National key scientific research projects (2012CB966604), the National Natural Science Fund (81125014, 81101356, 81201395, 31000436), Zhejiang province public welfare fund (2012C3112), Zhejiang Provincial Natural Science Foundation of China (LY13C100001), Postdoctoral Foundation of China (2013M531465), as well as sponsored by Regenerative Medicine in Innovative Medical Subjects of Zhejiang Province and Zhejiang Provincial Program for the Cultivation of High-level Innovative Health talents. The authors thank Mr. Hanmin Chen for SEM imaging, the Imaging Center of Zhejiang University School of Medicine for technical assistance and Mrs. Shufang Zhang for technical assistance.
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