Sage Journals: Discover world-class research

Abstract

This study examined how the difference in pore size of porous scaffolds affected the in vitro chondrogenic differentiation of seeded adipose stem cells (ASCs) and the in vivo cartilage repair of ASC/scaffold construct. ASCs were isolated from 18 rabbits and seeded in a porous poly (ε-caprolactone) (PCL) scaffold with different pore sizes (100, 200, 400 μm). The ASCs underwent in vitro chondrogenic induction under TGF-β₂ and BMP-7 for 21 days before analysis. The ASC/scaffold construct was also implanted on the osteochondral defect created on the distal femur of the same rabbits, and the quality of cartilage regeneration was analyzed after 8 weeks. At day 21, the ASCs proliferated and spread on the surface of the scaffolds with a pore size 100 and 200 μm, whereas there were many lumps of conglomerated ASCs on those with a pore size of 400 μm. The DNA content was significantly lower in the scaffold with a pore size of 400 μm than in that with a pore size of 100 or 200 μm. Proteoglycan production was significantly greater in the scaffold with a pore size of 400 and 200 μm than in that with a pore size of 100 μm. The chondrogenic marker gene expression including SOX9 and COL2A1 was greatest in the scaffold with a pore size of 400 μm followed by 200 μm. Immunofluorescent imaging showed that, while SOX9 was localized to nucleus, type II collagen was observed on the cytoplasm and secreted matrix around the cells most abundantly in the scaffold with a pore size of 400 μm followed by 200 μm. The gross and histological findings from the osteochondral defects showed that the cartilage repair was better in the scaffold with a pore size of 400 and 200 μm than in that with a pore size of 100 μm.

Keywords

Adipose stem cells (ASCs)Chondrogenesis Cartilage regeneration Pore size Porous scaffold

Introduction

Articular cartilage (AC) has limited potential for regeneration. Therefore, injuries to the AC do not heal spontaneously in adults, progressing into osteoarthritis (4,5). To promote cartilage repair in chondral defects, surgical procedures have been devised to mobilize stem cells from the bone marrow to AC defects, but they have not been successful in producing durable hyaline cartilage (1,8,22,26). Recently, tissue engineering approaches have been developed to restore functional cartilage (7). For cartilage tissue engineering, a three-dimensional (3D) porous scaffold provides a useful option that accommodates the implanted cells and guides new tissue formation (32). Synthetic biodegradable polymer scaffolds have advantages over natural scaffolds in tissue engineering because their structure and degradation time can be controlled according to the need of the specific tissue to be regenerated (24).

As a source of cells for cartilage tissue engineering, autologous chondrocytes were first tried and found to be useful for the purpose (3). However, disadvantages such as the morbidity of donor sites and the dedifferentiation of proliferated chondrocytes pose a limitations to the wide application of autologous chondrocytes (6,19). Recently, adipose stem cells (ASCs) have been considered good candidates for tissue engineering due to their multilineage potential (21,25,31,35,36). The abundance and easy availability of ASCs offer a huge advantage as a cell source for cartilage tissue engineering despite their lower chondrogenic potential when compared to mesenchymal stem cells (MSCs) from bone marrow (11,13). ASCs can achieve chondrogenesis comparable to bone marrow MSCs using a combination of transforming growth factor (TGF)-β₂ and bone morphogenetic proteins (BMPs) or with high dose of growth factors (16,17).

The factors that can influence tissue regeneration in a scaffold include the pore size, total porosity, pore shape, pore interconnectivity, material surface chemistry, effective scaffold degradability, and scaffold stiffness (32). Of these, the pore structure is the crucial characteristic. Pores must be interconnected to allow cell growth, migration, and nutrient flow (34). When the pore size is too small, cell migration, diffusion of nutrients, and removal of waste are limited, resulting in the death of implanted cells within the scaffold. Conversely, if the pores are too large, the surface area available for cell adhesion decreases and the mechanical strength of the scaffolds deteriorates with increasing void volume (14,33). Cells can also detect subtle changes in the extracellular matrix (ECM) that can affect their behavior during differentiation into a designated tissue as well as attachment and infiltration within biological scaffolds (2,10). Therefore, determination of the optimal pore size that can balance these variables is very important in designing a specific scaffold for tissue regeneration (14). Several groups reported different optimal pore sizes for chondrogenesis from chondrocytes (28,29,32), while there is a paucity of information on the optimal pore size for ASCs (27).

To investigate the pure effect of pore size on the behavior of seeded cells, it is important to exclude experimental artifacts due to the low repeatability of the pore size distribution. A scaffold fabricated with a pore size gradient can provide a powerful tool for this purpose. This study employed poly (ε-caprolactone) (PCL) cylindrical scaffolds with a pore size gradient fabricated using a modified centrifugation method, which were reported in previous investigations (27,28). PCL is one of the most widely used biodegradable polyesters for medical applications owing to its biocompatibility, biodegradability, and flexibility. A previous study demonstrated in vitro that PCL-based porous scaffolds can be a useful carrier for MSCs in cartilage tissue engineering (18). Although pore size of 370–400 μm was found to provide a more favorable environment for the chondrogenic differentiation of hASCs seeded in PCL scaffolds from a preceding study (27), the effects of the different pore size on the detailed morphological and functional changes in the seeded ASCs were not investigated, nor were the results of the in vivo implantation of these constructs. Therefore, the purpose of this study was to examine the influence of pore size on the in vitro appearance and chondrogenic differentiation of ASCs seeded in a porous PCL scaffold and on the in vivo cartilage repair of ASC/scaffold construct in a rabbit osteochondral defect model.

Materials and Methods

Isolation and Expansion of ASCs From the Rabbits

The animal experiments were approved by the Animal Research and Care Committee of our institution. Adipose tissue was obtained from the inguinal area of eighteen 8-week-old male rabbits (New Zealand White; Orientbio, Seongnam, Korea), weighing 3.0–3.5 kg as described in the previous study (12). Briefly, the rabbits were anesthetized with zoletil (40 mg/kg) and xylazine (10 mg/ kg). A 5-cm incision was made over the left inguinal area. The fatty tissue was exposed and excised. Adipose tissue was diced finely, washed three times with phosphate-buffered saline (PBS; Invitrogen, Grand Island, NY), digested with 1.5 mg/ml of collagenase (Invitrogen), and filtered through a 100-μm nylon mesh. The erythrocytes were removed using an erythrocyte lysis buffer. The remaining cells were placed in culture flasks and cultured in Dulbecco's modified Eagle's medium (DMEM)/Ham F-12 (Invitrogen) supplemented with 10% fetal bovine serum (FBS; Invitrogen) and 1% antibiotics (Invitrogen). The cultures were maintained at 37°C for 48 h in a humidified atmosphere containing 5% carbon dioxide (CO₂). During the expansion period, the medium was replaced twice per week. When the cells reached 80% confluence, they were detached from the culture dishes using 0.25% trypsin (Invitrogen) containing 1 mM ethylene diamine tetraacetic acid (EDTA), washed with PBS, counted, and replated. After culturing through passage 2, the cells were suspended in a cryopreservation medium containing 90% FBS and 10% dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO).

Fabrication of Pore Size Gradient PCL Scaffolds

The PCL cylindrical scaffolds with a pore size gradient along the longitudinal direction were fabricated by a modification of the centrifugation method (27,28). Briefly, hot PCL solution [(Sigma-Aldrich) 10 wt % (in tetraglycol), 90°C] was dropped slowly into water (nonsolvent for PCL) at room temperature with vigorous agitation using a homogenizer (20,000 rpm; HG-3000, SMT, Tokyo, Japan) to prepare the fibril-like PCLs, washed in excess water, and freeze-dried. The obtained fibril-like PCLs were resuspended in a cold Pluronic F127 solution ([BASF, USA] 20 wt %, ~4°C) to prepare a PCL/Pluronic F127 solution, 1:50 ratio (w/v). The solution was poured into a polypropylene cylindrical mold (inner diameter, 7 mm; height, 10 cm), and the mold was then centrifuged (3,000 rpm) for 5 min. During centrifugation, the fibril-like PCLs accumulated in the cylindrical mold with a density gradient of the PCLs (pore size gradient) that increase with a gradual increase in centrifugal force along the cylindrical axis. The pore size gradient PCL scaffold was eventually obtained using a fibril-bonding process. For this, the pore size gradient PCL prescaffold in a Pluronic F127 solution was stored at approximately 25°C for 1 h to induce the gelation of Pluronic F127 solution (sol–gel transition temperature of 20 wt % Pluronic F127, ~20°C). Then the prescaffold was heat-treated close to PCL's melting temperature (60°C) for 30 min to bond the accumulated PCL fibrils to each other and, thus, to prevent the disentanglement of fibrous structures in the cell culture medium or in the body. After the heat treatment, Pluronic F127 was washed out with excess water at 4°C overnight, and the scaffold was freeze-dried. The prepared PCL cylindrical scaffold was sectioned in a longitudinal direction to characterize them in terms of the scaffold pore sizes (section thickness, 2.0 mm). The sections with a pore size of 100 (mean ± SD: 101.5 ± 5.0), 200 (mean ± SD: 205.6 ± 14.0) and 400 (mean ± SD: 388.3 ± 9.2) μm were prepared for the in vitro and in vivo studies.

Seeding of ASCs Into PCL Scaffolds and In Vitro Chondrogenic Culture

The passage 3 cells were suspended in chondrogenic medium [CM: DMEM/F-12 supplemented with 1% ITS (insulin–transferrin–selenium; Invitrogen), 10⁻⁷ M dexamethasone (Sigma-Aldrich), 50 mM ascorbate-2-phosphate (Sigma-Aldrich), 50 mM l-proline (Sigma-Aldrich), and 1 mM sodium pyruvate (Sigma-Aldrich)] at a concentration of 5 × 10⁵ cell/40 ml. The cells were seeded into the PCL scaffolds by pipetting the cell suspension onto each side of the scaffold with a 4-h incubation period for each side. The ASC-seeded scaffolds were cultured in CM containing 5 ng/ml of transforming growth factor (TGF)-β₂ (R&D Systems, Inc., Minneapolis, MN) and 100 ng/ml of bone morphogenetic protein (BMP)-7 (R&D Systems) (12). The medium was changed every 3 days during the culture period. After 21 days, each ASC-seeded scaffold was homogenized under liquid nitrogen using a mortar and pestle for the biochemical assay and real-time PCR. The samples were also analyzed for DNA and glycosaminoglycan (GAG) 4 h after seeding at day 0 to provide a control.

Biochemical Assays for DNA and GAG Quantification

The DNA content was determined using the Quant-I T^™ dsDNA assay kit and Qubit Fluorometer system (Invitrogen). GAG production was determined using a Blyscan kit (Biocolor, Carrickfergus, UK) according to the manufacturer's instructions. The GAG content was determined using a standard curve drawn using standard solutions containing chondroitin 4-sulfate from the bovine trachea. The absorbance was measured at 656 nm in a Spectra Max plus 384 (Molecular Devices, Sunnyvale, CA). The GAG levels are expressed as micrograms of GAG per microgram of DNA. The DNA and GAG assays were conducted in triplicate for each sample.

Real-Time PCR

RNA extraction was performed using Trizol (Invitrogen) according to the manufacturer's instructions. The yield and purity of the extracted RNA was determined using the Quant-I T^™ RNA assay kit and Qubit Fluorometer system (Invitrogen). After the DNAse I treatment, the total RNA was reverse-transcribed with Multiscribe reverse transcriptase oligo (dT) primer (Invitrogen) in a 40-μl reaction volume, according to the manufacturer's instructions. All the PCR reactions were performed using a LightCycler 480 system® (Roche Diagnostics, Mannheim, Germany) in standard 10-μl reactions. The following primers were used: β-actin (forward, 5′-AGCAGATGTGGATCAGCAAG-3′; reverse, 5′-GTTTCGTCGAGAGAGGGTGT-3′), collagen type II, α1 [COL2A1] (forward, 5′-TGAGAA GGGACTTCCTGGAG-3′; reverse, 5′-TCCTGTCTCAC CATCTTTGC-3′), COL10A1 (forward, 5′-TGCATGTGA AAGGGACTCAT-3′; reverse, 5′-CCTGATCCAGGTA GCCCTTA-3′), and sex-determining region Y Box 9 [SOX9] (forward, 5′-ACGTCATCTCCAACATCGAG-3′; reverse, 5′-CCTTAACTGCCGGTGTAGGT). The relative normalization ratio of the PCR products derived from each target gene was calculated using software of the LightCycler System. All experiments were performed in triplicate for each sample.

Scanning Electron Microscopy (SEM) Images of the ASC/Scaffold Construct

After 7 and 21 days of in vitro culture, the scaffolds were fixed with 2.5% glutaraldehyde (Sigma-Aldrich) for 24 h at room temperature. After thorough washing with PBS, the cells adhered to the scaffolds were dehydrated in a graded series of ethanol and allowed to dry on a clean bench at room temperature. The surface and cross-section of the cell-adhered scaffolds were observed by SEM (Model Jsm 5410lv scanning microscope, Jeol, Tokyo, Japan).

Histological Analysis of the ASC/Scaffold Construct

After 21 days of in vitro culture, the ASC/scaffold construct were fixed in a 4% paraformaldehyde solution for 4 h, dehydrated with 100% and 95% ethanol, washed with xylene, and embedded in paraffin. Three-micrometer-thick sections were cut from the paraffin block and coated onto glass slides. Hematoxylin and eosin (H&E) and safranin-O staining for proteoglycan were then performed. For safranin-O staining, the specimen was deparaffinized with xylene and ethanol. Aqueous safranin-O (0.1%) was applied for 5 min and mounted.

Immunofluorescent Staining

The sections were deparaffinized, hydrated, and rinsed in several changes of distilled water. Subsequently, the sections were blocked with 5% normal donkey serum (NDS) and 0.1% Triton X-100 in PBS at room temperature for 1 h. The following primary antibodies were applied overnight at 4°C: rabbit polyclonal antibodies to type X collagen (1:100, Abcam, Cambridge, UK), SOX9 (1:100, Santa Cruz Biotechnology, Santa Cruz, CA), and mouse monoclonal antibody to type II collagen (1:100, Santa Cruz). The appropriate fluorescence-tagged secondary antibodies (R&D Systems) were used for visualization. The stained samples were mounted in VECTASHIELD^® with 4′,6-diamidino-2-phenylindole (DAPI) mounting medium (Vector Laboratories, Burlingame, CA) and photographed using an epifluorescence microscope (Leica, Wezlar, Germany). Measurement of the fluorescence intensity was calculated using the Leica Application Suite (LAS) image analysis package.

In Vivo Implantation of ASC/Scaffold Construct in the Osteochondral Defect

The rabbits were anesthetized in the same manner as described in a previous section, and the right legs were prepared aseptically. The knee joint was exposed by a medial parapatellar incision, and the trochlear groove was exposed by a lateral dislocation of the patella. A trephine drill with an outer diameter of 3 mm was used to create the osteochondral defects (diameter, 6 mm; depth, 3 mm) in the trochlear groove of the femur. The ASC/scaffold construct, which was prepared in the same way as in the in vitro study and cultured for 21 days, was implanted in the osteochondral defect created in the same rabbit from which the ASCs had been obtained. Six rabbits were allocated to each pore size (100, 200, and 400 μm). After implantation of the scaffolds, the patella was repositioned and the capsule was repaired using 4–0 nylon sutures. The rabbits were allowed to feed freely in their cage immediately after operation without immobilization.

Eight weeks after implantation, the rabbits were euthanized using an injected overdose of pentobarbital, and the defects were examined grossly and using the ICRS (International Cartilage Repair Society) macroscopic score, which evaluates the degree of defect repair, integration to the border zone, and macroscopic appearance (30). The distal femur was resected and fixed in a 4% paraformaldehyde solution for 7 days, after which it was decalcified and embedded in paraffin. The sections were made for H&E and safranin-O staining.

Statistical Analysis

Mann–Whitney U test was used to determine the inter-group differences in the scores. We have chosen not to correct for multiple comparisons. A value of p < 0.05 was considered significant.

Results

SEM and Histological Finding of ASCs Seeded in PCL Scaffolds of Different Pore Size After 7 and 21 Days of In Vitro Chondrogenic Culture

SEM was performed to investigate the cell–scaffold interaction between the ASCs and PCL scaffolds of different pore sizes. At day 7, the ASCs began to adhere to the scaffold surface. There was no remarkable difference between the scaffolds with different pore sizes. At day 21, the ASCs proliferated and spread over the scaffold surfaced with pore sizes of 100 and 200 μm, whereas there were many lumps of conglomerated ASCs in the scaffolds with pore size 400 μm (Fig. 1A). The tendency was also confirmed from H&E staining of the ASC/ scaffold construct. The ASCs were spread more evenly in the scaffolds with pore sizes of 100 and 200 μm than those with pore sizes of 400 μm (Fig. 1B).

Figure 1.

Scanning electron microscopy (SEM) and histological findings of the adipose stem cells (ASCs) seeded in poly (ε-caprolactone) (PCL) scaffolds with different pore sizes after 7 and 21 days of chondrogenic in vitro culture. At day 7, no remarkable difference was observed between the scaffolds with different pore sizes. At day 21, the ASCs proliferated and spread on the surface of the scaffolds with pore sizes of 100 and 200 μm, while there were many lumps of conglomerated ASCs on the scaffold with a pore size of 400 μm (A). The findings from H&E staining also confirmed that the ASCs were spread more evenly in the scaffolds with a pore size of 100 and 200 μm than those with a pore size of 400 μm (B).

DNA and GAG Synthesis in the ASC/PCL Scaffolds of Different Pore Size After 21 Days of In Vitro Chondrogenic Culture

The DNA content was examined from each ASC/ scaffold construct to indirectly confirm cell number, and GAG contents were also measured to assess proteoglycan synthesis.

At day 0, there was no difference in the DNA contents or GAG contents depending on pore sizes of the scaffold. After 21 days of in vitro culture, the DNA content of the ASCs seeded in the scaffold with pore sizes of 100 and 200 μm was similar. The DNA content was significantly lower in ASCs seeded in the scaffold with a pore size of 400 μm than in those with a pore size of 100 and 200 μm, by 12% and 14%, respectively (p = 0.037) (Fig. 2A). The GAG content normalized by the DNA content in ASCs was significantly greater in the scaffolds with a pore size of 400 and 200 μm than in those with a pore size of 100 μm, by 91% and 32%, respectively, at day 21 (p = 0.037) (Fig. 2B). Proteoglycan synthesis was also confirmed by safranin-O staining. ASCs that underwent chondrogenic differentiation in the PCL scaffold with a larger pore size showed greater metachromatic staining in the cytoplasm and matrix (Fig. 2C).

Figure 2.

DNA and glycosaminoglycan (GAG) synthesis in the ASC/PCL scaffolds of different pore sizes after 21 days of chondrogenic in vitro culture. The DNA content of the ASCs seeded in the scaffold with pore sizes of 100 and 200 μm was similar, while it was significantly lower in the scaffold with a pore size of 400 μm at day 21 (A). The GAG content normalized by the DNA content in ASCs was significantly greater in the scaffolds with pore sizes of 400 and 200 μm than in the scaffolds with a pore size of 100 μm at day 21 (B). Proteoglycan synthesis was also confirmed by safranin-O staining as indicated by arrows (C). n = 3, ∗p < 0.05.

Chondrogenic Marker Expression in the ASC/PCL Scaffolds of Different Pore Size After 21 Days of In Vitro Chondrogenic Culture

After 21 days of in vitro culture, the ASCs that underwent chondrogenic differentiation in the PCL scaffold with different pore sizes were analyzed for chondrogenic marker gene expression. SOX9 mRNA, the master transcription factor for chondrogenic differentiation, was 71% higher in the ASCs seeded in the scaffold with a pore size of 400 μm and 21% greater in that with a pore size of 200 μm than in those with a pore size of 100 μm (p = 0.037). COL2A1 mRNA expression, a marker of cartilaginous tissue, was 2.4-fold higher in the ASCs seeded in the scaffold with a pore size of 400 μm and 20% greater in that with a pore size of 200 μm than in those with a pore size of 100 μm (p = 0.037). There was no significant difference in COL10A1 mRNA expression, a marker of hypertrophy (Fig. 3A).

Figure 3.

Chondrogenic marker expression in the ASC/PCL scaffolds with different pore sizes after 21 days of chondrogenic in vitro culture. Sex-determining region Y box 9 (SOX9) mRNA and collagen type II α1 (COL2A1) mRNA expression increased with larger pore size, while there was no significant difference in COL10A1 mRNA expression, the marker of hypertrophy (A). The images from immunofluorescence staining show that, while SOX9 was localized to the nucleus, type II collagen was observed on the cytoplasm and secreted matrix around the cells (magnified insets on the left lower corner of merged panels, original magnification of insets: 3x). Type X collagen was found in small amounts on the cytoplasm (B). n = 3, ∗p < 0.05. NS, not significant.

The protein expression of SOX9, type II collagen, and type X collagen was also investigated using immunofluorescent imaging. While SOX9 was localized to nucleus, type II collagen was found on the cytoplasm and secreted matrix around the cells. Extracellular type II collagen was found most abundantly in the scaffold with a pore size of 400 μm followed by 200 μm. Type X collagen was found in only a few cells from all three pore sizes. Conglomerated ASCs were also noted in the PCL scaffolds with a pore size of 400 μm (Fig. 3B).

Gross and Histologic Findings From Rabbit Osteochondral Defects Implanted with the ASC/PCL Scaffolds of Different Pore Size for 8 Weeks

To examine the effect of the different pore size on cartilage regeneration by the ASC/scaffold construct, the construct was implanted on the osteochondral defect created on the patellar groove of the same rabbits. The rabbits were sacrificed 8 weeks after implantation. Although there were variation among individual rabbits, the gross appearance of osteochondral lesions and ICRS macroscopic scores were better in the scaffolds with a pore size of 200 μm (p = 0.036) and 400 μm (p = 0.045) than in those with a pore size of 100 μm (Fig. 4A, B). Histological assessment also demonstrated mixed hyaline cartilage-like and fibrocartilaginous tissue as well as the reconstitution of subchondral bone in the ASC/scaffold construct with a pore size of 200 and 400 μm, whereas only fibrocartilaginous or denuded surfaces were found in most ASC/scaffold constructs with a pore size of 100 μm (Fig. 4C).

Figure 4.

Gross and histological findings from rabbit osteochondral defects implanted with the ASC/PCL scaffolds with different pore size for 8 weeks. The gross appearance of the osteochondral lesion shows better quality of regenerated tissue in the osteochondral defect (A) (indicated by black arrows) and the International Cartilage Repair Society (ICRS) macroscopic scores were higher in the scaffolds with pore sizes of 200 and 400 μm than in the scaffold with a pore size of 100 μm (B). Histological assessment also demonstrates mixed hyaline cartilage-like and fibrocartilaginous tissue as well as reconstitution of subchondral bone in the ASC/scaffold construct with a pore size of 200 and 400 μm while only fibrocartilaginous or denuded surfaces are found in most ASC/scaffold constructs with a pore size of 100 μm. The margins of defects are indicated by the black arrows (C). n = 6, ∗p < 0.05. NS, not significant.

Discussion

This study provided detailed information on the cell–scaffold interaction including morphological and functional changes in the ASC/PCL scaffold construct with a different pore size. The overall in vitro findings suggested that a pore size of 200 μm had an advantage in cell proliferation and cell–scaffold interaction while a pore size of 400 μm was better in proteoglycan production and chondrogenic marker expression. On the other hand, the implantation study suggested that neither pore size was superior to one another.

Regarding proliferation, scaffolds with a smaller pore size initially allow greater cell attachment and thus enhance proliferation, even though the diffusion of nutrients and removal of waste can be hampered (14). The largest cell number of chondrocytes was observed in the chitosan-based scaffolds with the smallest pore size (100 μm) after 7 and 14 days of culture, whereas the scaffold with a pore size of 200 μm had the highest cell number after 28 days of culture (32). The poly (urethane urea) scaffold with a pore size of 150–300 μm had a higher number of chondrocytes after 28 days of in vitro culture than those with either <150 μm or 300–500 μm (29). In contrast, Oh et al., using the same scaffold as in the present study and human ASCs, found that the scaffold with a pore size of 90–105 μm showed the greatest number of ASCs after 21 days of in vitro culture, compared those with larger pore sizes, that is, 190–220, 300–329, and 370–400 μm (28). These results suggest that there is a certain optimum range of pore size for proliferation, which is 100–200 μm albeit varying moderately with the cell types and the material used in the scaffold. The present study, using rabbit ASCs, also showed that the number of cells were higher in the scaffolds with smaller pore sizes (100 and 200 μm) than in those with a larger pore size (400 μm). These scaffolds also showed a better interaction with the seeded ASCs than that with a large pore size.

There are also conflicting reports on the appropriate pore size for effective chondrogenesis. The effect of the pore size can be also influenced by the surface modification such as hydrophilization (23). A smaller pore size (<150 μm) in a hydrophobic poly (urethane urea) supported the deposition of hyaline cartilage-specific matrix components from seeded chondrocytes (29), whereas GAG deposition and type II collagen expression in hydrophilic chitosan-based hyaluronic acid hybrid polymer scaffolds were greater with a larger pore size, that is, 400 μm compared with size of 200 and 100 μm (32). Although lower oxygen tension in the scaffolds with smaller pores may give some chondrogenic stimulus (9,20), the larger pores offers an environment for a more effective nutrient supply and waste removal, which allows greater anabolic activity (28). While previous studies that compared the effect of the pore size on cartilage matrix production used mature chondrocytes, the undifferentiated stem cells as used in the present study may yield different results, as demonstrated by a recent study that observed the opposite effect of increased permeability on chondrocytes and bone marrow stem cells (15). Nevertheless, the present study, which used ASCs in PCL scaffold hydrophilized by Pluronic F127, produced similar results to those reported by Yamane et al., who used chondrocytes (32). Proteoglycan and chondrogenic markers were clearly higher in scaffold with a larger pore size, that is, 400 μm, even though it was not translated into better results in the in vivo implantation study.

There are several points in the results of this study that should be interpreted with care. First, the effect of the pore size on the proteoglycan synthesis and gene expression was not as remarkable as seen in other treatments, such as growth factors. Although the different pore sizes definitely affects chondrogenesis of ASCs seeded in the scaffold, the magnitude of the chondrogenic effect may not be compared with that of TGF-β or BMPs, which induced a several-fold larger increase in the expression of the chondrogenic markers (16,17). Second, implantation time was rather short. A longer implantation time could have resulted in improved quality of repair, considering the longer biodegradation time of PCL. Despite these shortcomings, the present study provided an in vitro and in vivo model to investigate the pure effect of pore size on the chondrogenesis from ASCs, which may be used in other cells and scaffold systems.

In conclusion, while the scaffold with a pore size of 200 μm showed better performance in terms of cell proliferation and cell–scaffold interaction, the scaffold with a pore size of 400 μm excelled in enhancing chondrogenic differentiation. The scaffolds with pore sizes of 200 and 400 μm had similar outcomes in the in vivo implantation study. Both of them may be utilized for cartilage repair with ASCs.

Footnotes

Acknowledgment

This work was supported by grants from the National Research Foundation of Korea (2009-0092196 and 2008-314-D00515). The authors declare no conflicts of interest.

References

Beiser

I. H.

Kanat

I. O.

Subchondral bone drilling: A treatment for cartilage defects. J. Foot Surg. 29: 595–601; 1990.

Boyan

B. D.

Hummert

T. W.

Dean

D. D.

Schwartz

Role of material surfaces in regulating bone and cartilage cell response. Biomaterials 17: 137–146; 1996.

Brittberg

Lindahl

Nilsson

Ohlsson

Isaksson

Peterson

Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N. Engl. J. Med. 331: 889–895; 1994.

Buckwalter

J. A.

Mankin

H. J.

Articular cartilage: Degeneration and osteoarthritis, repair, regeneration, and transplantation. Instr. Course Lect. 47: 487–504; 1998.

Buckwalter

J. A.

Mankin

H. J.

Grodzinsky

A. J.

Articular cartilage and osteoarthritis. Instr. Course Lect. 54: 465–480; 2005.

Caplan

A. I.

Review: Mesenchymal stem cells: Cell-based reconstructive therapy in orthopedics. Tissue Eng. 11: 1198–1211; 2005.

Caplan

A. I.

Goldberg

V. M.

Principles of tissue engineered regeneration of skeletal tissues. Clin. Orthop. Relat. Res. 367(Suppl): S12–S16; 1999.

Childers

J. C.

Jr. Ellwood

S. C.

Partial chondrectomy and subchondral bone drilling for chondromalacia. Clin. Orthop. Relat. Res. 144: 114–120; 1979.

Domm

Schunke

Steinhagen

Freitag

Kurz

Influence of various alginate brands on the redifferentiation of dedifferentiated bovine articular chondrocytes in alginate bead culture under high and low oxygen tension. Tissue Eng. 10: 1796–1805; 2004.

10.

Harley

B. A.

Kim

H. D.

Zaman

M. H.

Yannas

I. V.

Lauffenburger

D. A.

Gibson

L. J.

Microarchitecture of three-dimensional scaffolds influences cell migration behavior via junction interactions. Biophys. J. 95: 4013–4024; 2008.

11.

Huang

J. I.

Zuk

P. A.

Jones

N. F.

Zhu

Lorenz

H. P.

Hedrick

M. H.

Benhaim

Chondrogenic potential of multipotential cells from human adipose tissue. Plast. Reconstr. Surg. 113: 585–594; 2004.

12.

G. I.

Lee

J. H.

Repair of osteochondral defects with adipose stem cells and a dual growth factor-releasing scaffold in rabbits. J. Biomed. Mater. Res. B Appl. Biomater. 92: 552–560; 2010.

13.

G. I.

Shin

Y. W.

Lee

K. B.

Do adipose tissue-derived mesenchymal stem cells have the same osteogenic and chondrogenic potential as bone marrow-derived cells?

Osteoarthritis Cartilage 13: 845–853; 2005.

14.

Karageorgiou

Kaplan

Porosity of 3D biomaterial scaffolds and osteogenesis. Biomaterials 26: 5474–5491; 2005.

15.

Kemppainen

J. M.

Hollister

S. J.

Differential effects of designed scaffold permeability on chondrogenesis by chondrocytes and bone marrow stromal cells. Biomaterials 31: 279–287; 2010.

16.

Kim

H. J.

G. I.

Chondrogenic differentiation of adipose tissue-derived mesenchymal stem cells: Greater doses of growth factor are necessary. J. Orthop. Res. 27: 612–619; 2009.

17.

Kim

H. J.

G. I.

Combination of transforming growth factor-beta(2) and bone morphogenetic protein 7 enhances chondrogenesis from adipose tissue-derived mesenchymal stem cells. Tissue Eng. Part A 15: 1543–1551; 2009.

18.

Kim

H. J.

Lee

J. H.

G. I.

Chondrogenesis using mesenchymal stem cells and PCL scaffolds. J. Biomed. Mater. Res. A 92: 659–666; 2010.

19.

Lefebvre

Peeters-Joris

Vaes

Production of collagens, collagenase and collagenase inhibitor during the dedifferentiation of articular chondrocytes by serial subcultures. Biochim. Biophys. Acta 1051: 266–275; 1990.

20.

Malda

van Blitterswijk

C. A.

van Geffen

Martens

D. E.

Tramper

Riesle

Low oxygen tension stimulates the redifferentiation of dedifferentiated adult human nasal chondrocytes. Osteoarthritis Cartilage 12: 306–313; 2004.

21.

Merceron

Portron

Masson

Lesoeur

Fellah

B. H.

Gauthier

Geffroy

Weiss

Guicheux

Vinatier

The effect of two and three dimensional cell culture on the chondrogenic potential of human adipose-derived mesenchymal stem cells after subcutaneous transplantation with an injectable hydrogel. Cell Transplant. 20: 1575–1588; 2011.

22.

Minas

Nehrer

Current concepts in the treatment of articular cartilage defects. Orthopedics 20: 525–538; 1997.

23.

Miot

Woodfield

Daniels

A. U.

Suetterlin

Peterschmitt

Heberer

van Blitterswijk

C. A.

Riesle

Martin

Effects of scaffold composition and architecture on human nasal chondrocyte redifferentiation and cartilaginous matrix deposition. Biomaterials 26: 2479–2489; 2005.

24.

Mukherjee

D. P.

Smith

D. F.

Rogers

S. H.

Emmanual

J. E.

Jadin

K. D.

Hayes

B. K.

Effect of 3D-microstructure of bioabsorbable PGA: TMC scaffolds on the growth of chondrogenic cells. J. Biomed. Mater. Res. B Appl. Biomater. 88: 92–102; 2009.

25.

Nathan

Das De

Thambyah

Fen

Goh

Lee

E. H.

Cell-based therapy in the repair of osteochondral defects: A novel use for adipose tissue. Tissue Eng. 9: 733–744; 2003.

26.

O'Driscoll

S. W.

The healing and regeneration of articular cartilage. J. Bone Joint Surg. Am. 80: 1795–1812; 1998.

27.

S. H.

Kim

T. H.

G. I.

Lee

J. H.

Investigation of pore size effect on chondrogenic differentiation of adipose stem cells using a pore size gradient scaffold. Biomacromolecules 11: 1948–1955; 2010.

28.

S. H.

Park

I. K.

Kim

J. M.

Lee

J. H.

In vitro and in vivo characteristics of PCL scaffolds with pore size gradient fabricated by a centrifugation method. Biomaterials 28: 1664–1671; 2007.

29.

Stenhamre

Nannmark

Lindahl

Gatenholm

Brittberg

Influence of pore size on the redifferentiation potential of human articular chondrocytes in poly(urethane urea) scaffolds. J. Tissue Eng. Regen. Med. 5: 578–588; 2011.

30.

van den Borne

M. P.

Raijmakers

N. J.

Vanlauwe

Victor

de Jong

S. N.

Bellemans

Saris

D. B.

International Cartilage Repair Society (ICRS) and Oswestry macroscopic cartilage evaluation scores validated for use in Autologous Chondrocyte Implantation (ACI) and micro-fracture. Osteoarthritis Cartilage 15: 1397–1402; 2007.

31.

Wei

Wang

Deng

Cartilage regeneration of adipose-derived stem cells in a hybrid scaffold from fibrin-modified PLGA. Cell Transplant. 18: 159–170; 2009.

32.

Yamane

Iwasaki

Kasahara

Harada

Majima

Monde

Nishimura

Minami

Effect of pore size on in vitro cartilage formation using chitosan-based hyaluronic acid hybrid polymer fibers. J. Biomed. Mater. Res. A 81: 586–593; 2007.

33.

Yannas

I. V.

Tissue regeneration by use of collagen-glycosaminoglycan copolymers. Clin. Mater. 9: 179–187; 1992.

34.

Zeltinger

Sherwood

J. K.

Graham

D. A.

Mueller

Griffith

L. G.

Effect of pore size and void fraction on cellular adhesion, proliferation, and matrix deposition. Tissue Eng. 7: 557–572; 2001.

35.

Zuk

P. A.

Zhu

Ashjian

De Ugarte

D. A.

Huang

J. I.

Mizuno

Alfonso

Z. C.

Fraser

J. K.

Benhaim

Hedrick

M. H.

Human adipose tissue is a source of multipotent stem cells. Mol. Biol. Cell 13: 4279–4295; 2002.

36.

Zuk

P. A.

Zhu

Mizuno

Huang

Futrell

J. W.

Katz

A. J.

Benhaim

Lorenz

H. P.

Hedrick

M. H.

Multilineage cells from human adipose tissue: Implications for cell-based therapies. Tissue Eng. 7: 211–228; 2001.

Chondrogenesis of Adipose Stem Cells in a Porous Polymer Scaffold: Influence of the Pore Size

Abstract

Keywords

Introduction

Materials and Methods

Isolation and Expansion of ASCs From the Rabbits

Fabrication of Pore Size Gradient PCL Scaffolds

Seeding of ASCs Into PCL Scaffolds and In Vitro Chondrogenic Culture

Biochemical Assays for DNA and GAG Quantification

Real-Time PCR

Scanning Electron Microscopy (SEM) Images of the ASC/Scaffold Construct

Histological Analysis of the ASC/Scaffold Construct

Immunofluorescent Staining

In Vivo Implantation of ASC/Scaffold Construct in the Osteochondral Defect

Statistical Analysis

Results

SEM and Histological Finding of ASCs Seeded in PCL Scaffolds of Different Pore Size After 7 and 21 Days of In Vitro Chondrogenic Culture

DNA and GAG Synthesis in the ASC/PCL Scaffolds of Different Pore Size After 21 Days of In Vitro Chondrogenic Culture

Chondrogenic Marker Expression in the ASC/PCL Scaffolds of Different Pore Size After 21 Days of In Vitro Chondrogenic Culture

Gross and Histologic Findings From Rabbit Osteochondral Defects Implanted with the ASC/PCL Scaffolds of Different Pore Size for 8 Weeks

Discussion

Footnotes

Acknowledgment

References