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Cartilage regeneration using adipose-derived stem cells and the controlled-released hybrid microspheres
Joint Bone Spine 77 (2010) 27–31
Original article
Cartilage regeneration using adipose-derived stem cells and the controlled-released hybrid microspheres Yisheng Han ∗ , Yiyong Wei , Shuseng Wang , Yang Song Department of orthopaedics, Xijing Hospital, The Fourth Military Medical University, West Road Changle, Xi’an, China
a r t i c l e
i n f o
Article history: Accepted 19 May 2009 Keywords: Adipose-derived stem cells Gelatin Chitosan Microspheres Cartilage regeneration
a b s t r a c t Objective: This study was to evaluate the effect of hybrid microspheres (MS) composed of gelatin transforming growth factor-beta (TGF-1)-loaded MS and chitosan MS on the enhancement of differentiation of adipose-derived stem cells (ASCs) into chondrocytes in pellet culture in vitro and the reparative capacity of pellet from ASCs and the hybrid MS-TGF used to repair cartilage defects in vivo. Methods: The morphology of the controlled-released MS was observed with scanning electron microscopy (SEM) and mechanical property was also tested in this study. In vitro TGF-1 release was evaluated by an enzyme-linked immunosorbent assay. The protein expression of Collagen II was tested by Western blot. In addition, a preliminary study on cartilage regeneration was also performed in vivo. Results: When chondrogenic differentiation of ASCs in both MS was evaluated, the protein expression of Collagen II became significantly increased for the hybrid MS-TGF, as compared with the gelatin MS-TGF. Mechanical result showed that the hybrid MS was superior to the gelatin MS. Observation of histology in vivo demonstrated that the pellet from ASCs and the hybrid MS-TGF promoted cartilage regeneration in the defects of articular cartilage much better than other groups. Conclusion: Our study demonstrated that the pellet from ASCs and the hybrid MS-TGF can provide an easy and effective way to construct the tissue engineered cartilage in vitro and in vivo. © 2009 Published by Elsevier Masson SAS on behalf of the Société Française de Rhumatologie.
1. Introduction As an avascular tissue with a very slow turnover at the cellular and molecular levels, articular cartilage has a limited capacity for self-repair when damaged as a result of trauma or degenerative diseases. Consequently, the requirement for new cartilage tissue to repair the damaged cartilage is a major concern. Over the past several decades, many solutions have been developed to restore the normal function of the injured cartilage including abrasion, drilling and microfracture [1–3]. Although all of these approaches have proved to be beneficial, none are believed to be optimal for repairing cartilage lesions. Cartilage tissue engineering provides a promising option for the repair of severe damage. The major cell source used to generate engineered cartilage tissue is stem cells. To date, adult stem cells have been identified in various tissues including bone marrow [4], muscle [5] and adipose tissue [6]. Studies have indicated that adult stem cells from adipose tissue contain multipotent progenitor cells, which have the potential to differentiate into osteogenic, chondrogenic, myogenic and neurogenic cells [6,7]. During in vitro expansion, adipose-derived stem cells (ASCs) can maintain a stable undifferentiated status, and
∗ Corresponding author. E-mail address: [email protected] (Y. Han).
pluripotency does not decrease in a growing body. In addition, because adipose tissue is obtained by extraction from the patient in a less invasive manner and provides a large quantity of autologous cells, ASCs represent a fascinating cell source for regenerative medicine. It has been reported that various kinds of growth factors (GFs) affect chondrocyte metabolism and chondrogenesis. Among GFs of potential interest is transforming growth factor-beta (TGF-). As the predominant isoform of TGF-, TGF-1 plays an important role in promoting chondrogenic differentiation in chondroblasts and in mesenchymal cells [8]. Although studies have demonstrated that TGF-1 added directly to the cell culture media induces chondrogenic differentiation of stem cells in vitro, this method decreased the efficacy of TGF-1. Moreover, a suitable concentration was not maintained over an extended time period. So the controlled-release of TGF-1 from a scaffold has become a topic of interest. Gelatin is a partial derivative of collagen. Studies have demonstrated that gelatin MS loaded with TGF-1 could promote chondrocyte growth and differentiation and could maintain the chondrogenic phenotype [9,10]. Moreover, the gelatin MS loaded with TGF-1 could retard the dedifferentiation of chondrocytes when the composite of cells and gelatin is implanted into the body [10]. However, the functional outcome of gelatin is often limited by its inherent poor mechanical stability. In order to increase chondrogenesis and the quality of tissue repair, the use of hybrid
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microspheres (MS) based on gelatin has recently drawn more attention. Chitosan is a natural polymer, which is structurally similar to various glycosaminoglycans (GAGs) found in articular cartilage and has been reported to be non-toxic, bio-absorbable and to promote would healing [11]. In addition, the study also demonstrated that the mechanical properties of gelatin blended with chitosan were significantly increased when compared to that of gelatin alone, which will promote cartilage regeneration [12]. In this study, we hypothesized that the hybrid TGF1-loaded MS, composed of the gelatin TGF1-loaded MS in combination with the chitosan MS, could provide superior biological effect on the differentiation of ASCs into chondrocytes in pellet culture in comparison with the gelatin MS loaded with TGF-1. To test this hypothesis, the controlled-released hybrid MS were fabricated, and then the mixture of ASCs and the controlled-released hybrid MS was centrifuged to form a pellet. Cell differentiation in the pellet of ASCs-the hybrid MS-TGF was determined and compared with these characteristics in the pellet of ASCs-the gelatin MS-TGF in vitro. In addition, the study is also to evaluate the reparative capacity of the pellet from ASCs and the hybrid MS-TGF used to repair cartilage defects in vivo.
2. Methods 2.1. Preparation of the gelatin MS-TGF and the hybrid MS-TGF The gelatin MS were prepared according to the reported method [9]. Briefly, 1.1 g of acidic gelatin (Sigma Co., St Louis, MO) was dissolved in 7 mL of double-distilled water (dd H2 O) at 53 ◦ C. The aqueous gelatin solution was added dropwise to 30 mL of olive oil and 0.2 mL of Tween 80 (Sigma Co.) while stirring at 500 rpm. The temperature of emulsion was then decreased to 0 ◦ C with constant stirring. After 12 min, 10 mL of chilled acetone (4 ◦ C) was further added. Then, the MS collected were washed with acetone to remove residual oil and cross-linked with 10 mM glutaraldehyde (GA) (Sigma Co.) at 4 ◦ C for 24 h. The cross-linked MS were washed with dd H2 O and counted using a hemocytometer. Then, 500 L of suspension containing 2 × 104 MS was placed in a 1.0 mL microcentrifuge tube for lyophilization. The dried MS (2 × 104 ) were loaded with 50 ng TGF-1 (Protec) by swelling in 5 L of TGF-1 solution (10 ng/L) at pH 7.4. The solution volume is below the MS’ theoretical equilibrium swelling volume to allow for complete TGF-1 absorption. The mixture was gently stirred with a pipette tip and then incubated at 4 ◦ C overnight for thorough swelling, followed by lyophilization. Chitosan MS were prepared according to the reported method [13]. Briefly, chitosan (120 mg) was dissolved in 2% aqueous acetic acid solution (4 ml) until the solution was transparent. The solution was added slowly to HCO-60 solution (5 w/v% in n-octanol) and emulsified at 13,500 rpm by using a homogenizer. After 20 min, 10 ml of TPP solution (10 w/v% in distilled water) was further added dropwise to stabilize the chitosan MS through the electrostatic interaction with TPP. The MS, precipitated in the mixture solvent, were repeatedly washed with excess amounts of isopropyl alcohol and water, and counted using a hemocytometer. Then, 500 L of suspension containing 2 × 104 MS were placed in a 1.0 mL microcentrifuge tube for lyophilization. To construct the hybrid MS-TGF, the gelatin MS-TGF and the chitosan MS (1:1) were crosslinked by 0.3% EDAC at 37 ◦ C for 1 h while being gently stirred. The hybrid MS-TGF was repeatedly washed with phosphate buffered saline (PBS) and then incubated at 4 ◦ C overnight by lyophilization. The morphology of the gelatin MS-TGF and the hybrid MS-TGF was observed with scanning electron microscopy (SEM).
2.2. Mechanical testing measurement Five hundred microlitres of suspension containing MS (the gelatin MS and hybrid MS respectively) were placed in the cylindrical mold for lyophilization, and then gelatin was added into the mold (200 L) for lyophilization to form the cylinder (height 3 mm and diameter 3 mm). Scaffolds were neutralized with alcohol and washed with PBS (pH = 7.4) for 30 min prior to testing (Instron 5569). Tests were performed in 37 ◦ C PBS at a rate of 5 mm/min. 2.3. In vitro TGF-ˇ1 release study In vitro, the gelatin MS-TGF and the hybrid MS-TGF (2 × 104 ) were dispersed into 5 mL of PBS and placed in a shaking water bath at 37 ◦ C, 135 rpm. After 0.5, 1, 3, 5, 7, 10, 14, 21 and 28 days, the suspension was centrifuged at 13,500 rpm to collect the supernatant for TGF-1 analysis. The PBS buffer was added afterward to keep the volume constant. The analysis was performed by an enzyme-linked immunosorbent assay kit (R&D Systems). 2.4. Isolation of adipose-derived stem cells and culture ASCs were isolated from the cervical adipose tissue of adult rabbits (age ≤ 4 months), using a method reported previously [14]. In brief, the tissue obtained was washed with equal volumes of PBS to remove red cells. The adipose tissue was minced finely using surgical scissors and the extracellular matrix was digested for 1 h at 37 ◦ C with 0.15% collagenase (type I; Sigma, St Louis, MO, USA) in PBS. Once digested, enzyme activity was neutralized with culture medium containing DMEM (Gibco, Paisley, UK), 10% FBS (Sijiqing Biological Engineering Materials Co., Hangzhou, China), penicillin (100 U/mL) and streptomycin (100 g/mL). The samples were filtered through a 500 m mesh filter to remove tissue debris. The cell suspension was centrifuged at 800 g to obtain a pellet and the pellet was resuspended in culture medium. The cells were seeded in 25-cm flasks (Corning-Costar, Acton, MA, USA) at density of 4 × 105 cells/cm3 and incubated at 37 ◦ C/5%CO2 . The medium was exchanged after 24 h and then three times a week. At a confluence of more than 80%, cells were detached with 0.25% trypsin and 0.1% EDTA. The cells were passaged five times prior to use. 2.5. Pellet culture Pellet culture was used according to a method reported previously [14]. Briefly, the mixture of ASCs (2 × 105 ) with the gelatin MS-TGF or the hybrid MS-TGF (2 × 104 ) was centrifuged at 500 g for 10 min in 15 mL polypropylene falcon tubes to form a cell pellet. Then, the pellets were cultured in 2 mL basal medium containing 1% FBS, 1 × insulin-transferrin-selenium (ITS) and antibiotics at 37 ◦ C, 5% CO2 , over a period of 2 weeks. The culture medium was changed every other day. 2.6. Histological analysis of pellet Samples were fixed for histological analysis with 4% paraformaldehyde in PBS, embedded in paraffin and sectioned using standard histochemical techniques. Slide sections were stained with H&E. 2.7. Western blot of Collagen II from pellet Protein extracts from chondrocytes, the composites of ASCs-the gelatin MS-TGF and the composites of ASCs-the hybrid MS-TGF were used for Western blot analysis. The protein concentration of the extracts was assessed using a Bradford assay kit. The optical density was measured at 595 nm using a spectrophotometer.
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The protein concentration was interpolated from a standard curve obtained from a serial dilution of bovine serum albumin at a concentration of 0.8 mg/ml. The cell extracts, normalized for total protein content, were resolved using polyacrylamide gel electrophoresis and electrophoretically transferred to a supported nitrocellulose membrane (Amersham, USA). Afterwards, the membranes were blocked in 5% non-fat milk tris-buffered saline tween (TBST) for 2 h, and incubated with primary antibodies, mouse monoclonal anti-collagen II (1:200, Chemicon, USA) at 4 ◦ C overnight. Membranes were then washed three times for 10 min each with TBST, followed by the addition of peroxidase-linked anti-mouse IgG (1:500) and enhanced chemiluminescence (ECL) visualization of the bands. 2.8. Repair of articular defects A cylindrical, full-thickness defect, thickness: 3 mm; diameter: 4 mm, was created using a hand drill with a 4-mm drill bit, penetrating the articular cartilage in the patellar groove of the distal femur in 18 rabbits (the ASCs from them had been prepared as mentioned above). The MS (the gelatin MS-TGF and the hybrid MSTGF, 1 × 105 ) containing ASCs (1 × 106 ) were centrifuged at 500 g for 10 min in the polypropylene falcon tubes to form cell pellets. The pellets were cultured at 37 ◦ C/5%CO2 for 24 h before transplantation experiment. The rabbits were operated on by injection of 2 mL/kg Sumianxin (Agricultural University, Changchun, China) into the muscle. The rabbits were classified into the following three groups: (i) the cartilage defect group (no insertion group), (ii) ASCs-the gelatin MS-TGF group, and (iii) ASCs-the hybrid MSTGF. All rabbits were allowed to move freely after surgery without plaster immobilization. The committee on animal experimentation of Fourth Military Medical University approved all experiments, which met the National Institute of Healthy (NIH) guidelines for the care and use of laboratory animals. Samples were harvested 12 weeks postoperatively. The distal parts of femurs were removed and fixed in 10% paraformaldehyde, decalcified in 10% EDTA and embedded in paraffin, sectioned at 6 m in thickness and stained with toluidine blue. Cartilage repair of the defects (six sections taken from each defect) was evaluated microsopically and scored according to a histological grading scale modified after Wakitani et al. [15] and Pineda et al. [16], consisting of five categories with a total score ranging from 0–20 points. 2.9. Statistical analysis All the data were expressed as the mean ± standard deviation (S.D.). The values in vitro study were analyzed using ANOVA-test. In vivo study histological scores among groups were analyzed with Mann-Whitney test and the Bonferroni method (n = 6). P-value difference less than 0.05 was considered to be statistically significant.
Fig. 1. Scanning electron microscopic images of the gelatin MS-TGF (A) and the hybrid MS-TGF (B) at the original magnification × 4000.
3.3. In vitro TGF-ˇ1 release study A parallel study was undertaken to examine the release kinetics of TGF-1 from the gelatin MS-TGF and the hybrid MS-TGF over 28 days. TGF-1 was released in a biphasic fashion characterized by a burst release at the initial 24 h, which continued until 72 h, after which a plateau was reached. The plateau was maintained until the end of the release period (4 weeks). A fast release on the first day was significantly higher (P < 0.05) for the hybrid MS-TGF (45.2%) when compared to the gelatin MS-TGF (35.9%). Then, the release increased steadily in both the gelatin MS-TGF and the hybrid MSTGF. By day 28, the cumulative release from the hybrid MS-TGF was 92% and it significantly exceeded (P < 0.05) the release from the gelatin MS (77%) (Fig. 3). 3.4. Histological analysis Histological examination using H&E staining indicated that a distinct cartilage-specific morphological appearance and structural characteristics such as lacunae are normally observed in both constructs (Fig. 4). In addition, an unequal distribution of the differentiated ASCs was evident and the differences of cell number were visible between the gelatin MS-TGF and the hybrid MS-TGF. 3.5. Western blot analysis for collagen II Collagen II is the most important component of extracellular matrix (ECM) in native articular cartilage, so its expression in the pellet of ASCs-the MS was tested to evaluate the differentiated status of ASCs. At day 14, the expression of Collagen II in ASCsthe hybrid MS-TGF was significantly higher than that in ASCs-the gelatin MS-TGF (P < 0.05) (Fig. 5). 3.6. In vivo histological evaluation Histological study (Fig. 6) showed that in ASCs-the hybrid MSTGF group, the defects were completely filled with reparative tissue
3. Results 3.1. Characterization of MS-TGF SEM images showed the morphology of the gelatin MS-TGF and the hybrid MS-TGF. Two kinds of MS-TGF were all spherical and had smooth surface (Fig. 1). 3.2. Mechanical properties of the gelatin microspheres (MS) and the hybrid MS Stress-strain curve showed that the stress value was obviously higher in the hybrid MS compared to the gelatin MS when the strain value was the same (Fig. 2).
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Fig. 2. Stress-strain curve of the gelatin microspheres (MS) and the hybrid MS.
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Fig. 6. Histological observation of cartilage repair at postoperative 12 weeks. A. The cartilage defect group. B. Adipose-derived stem cells (ASCs)-the gelatin MS-TGF group. C. ASCs-the hybrid MS-TGF group (toluidine blue staining) (× 100).
Fig. 3. Release profiles of TGF-1 from the gelatin MS-TGF and the hybrid MS-TGF. Data are expressed as mean ± standard deviation (n = 5). The statistical evaluation of the data is detailed in the text.
and the surface of reparative tissue was regular, and integration of the newly formed cartilage with host cartilage was the best among these groups. In addition, extensively metachromatic staining was showed. In ASCs-the gelatin MS-TGF group, the surface of reparative tissue was regular and the integration of donor with host adjacent cartilage was not very good, and the intensity of safranin O declined. The defects were filled with fibrous tissue in the cartilage defect group. The means in Wakitani’s score of the cell-containing MS group exhibited significantly better cartilage regeneration than those of the cartilage defect group at 12 weeks (Table 1). Compared with ASCs-the gelatin MS-TGF group, ASCs-the hybrid MS-TGF group showed much better regeneration of cartilage tissue. 4. Discussion
Fig. 4. H&E staining of adipose-derived stem cells (ASCs)-the gelatin MS-TGF (A × 400) and ASCs-the hybrid MS-TGF (B × 400) at day 14.
Fig. 5. The protein expression of Collagen II for adipose-derived stem cells (ASCs)the gelatin MS-TGF and ASCs-the hybrid MS-TGF at day 14. Data are expressed as mean ± standard deviation (n = 5). *statistically significant relative to ASCs-the gelatin MS-TGF (P < 0.05).
Pellet culture as a culture model results in the formation of three-dimensional structures and mimics precartilage condensations during embryonic development, cell-to-cell interactions among chondrocytes are known to be important in preventing dedifferentiation. Some researchers demonstrated that stem cells plated at high density could accelerate chondrogenic differentiation in vitro [17,18]. Our study had also demonstrated that pellet culture was an appropriate way to control chondrogenesis of ASCs [19]. To further enhance chondrogensis, a modified pellet was formed using ASCs and hybrid MS with controlled-released TGF1. In this study we selected two types of natural polymers to construct the hybrid MS. Gelatin was chosen as the base for the hybrid MS in this study for the following reasons: first, it contains an Arg-Gly-Asp (RGD)-like sequence that promotes cell adhesion and migration; second, it has low immunogenicity and low cost; finally, it can be degraded entirely in vivo and its degradation products are non-toxic [20]. In addition to the characteristics of gelatin mentioned above, it can also provide the release of GFs in a controlled fashion, which can serve to increase the in-growth and biosynthetic ability of cells. However, the use of gelatin for cartilage defect repair has limitations due to its inherent poor mechanical stability [21]. Mechanical property is a key factor in cartilage tissue engineering because the scaffolds should keep proper strength until the newly-formed tissue becomes mechanically competent after implantation. The result of our study showed that the mechanical property of the
Table 1 Results of histological grading (mean values ± standard deviation). n
Control ASCs-the gelatin MS-TGF ASCs-the hybrid MS-TGF
Scores on histologic grading scale
6 6 6
Cell morphology and matrix staining (0–8)
Surface regularity
Integration
Filling of defects
Total
(0–4)
Subchondral bone reconstruction (0–3)
(0–3)
(0–2)
7.8 ± 0.5 5.6 ± 0.3 4.8 ± 0.6
2.9 ± 0.3 1.9 ± 0.4 1.3 ± 0.4
1.8 ± 0.4 1.2 ± 0.4 0.9 ± 0.5
3.8 ± 0.2 1.5 ± 0.1 1.1 ± 0.6
2.7 ± 0.5 1.1 ± 0.1 0.8 ± 0.3
18.2 ± 0.7 9.8 ± 0.5# 7.5 ± 0.4*#
*P < 0.05 compared with ASCs-the gelatin MS-TGF group, #P < 0.05 compared with the cartilage defect group. a The scale has five categories assigning a total score ranging from 0 (best) to 20 (worst).
(0–20)a
Y. Han et al. / Joint Bone Spine 77 (2010) 27–31
hybrid MS was superior to that of the gelatin MS by the stressstrain curve. Based on the significant increase of the mechanical properties of gelatin blended with chitosan, hybrid MS made from gelatin containing TGF-1 and chitosan were fabricated. The in vitro release of TGF-1 was examined in this study. The study illustrated that there were relatively high release levels of TGF-1 from the hybrid MS when compared to the gelatin MS during the observed period. A possible mechanism is that the positive charge was increased by adding the chitosan MS to the gelatin MS-TGF-1, which resulted in the rapid release of TGF-1 due to electrostatic repulsion. The rapid release will increase the bioactive effect of TGF-1 on stem cells by influencing cell migration, proliferation and differentiation. We also demonstrated that the release profile of TGF-1 in both constructs showed a biphasic model with an initial burst followed by a plateau, which indicated an extended time course for complete release. The earlier burst release is likely caused by the rapid diffusion of TGF-1 located close to the surface of MS, as water uptake plays an important role in modulating the release of TGF-1 in the initial stage. Although the biological activity of TGF-1 was not quantified in this study, the chondrogenic differentiation of ASCs suggested that its activity was preserved. The ELISA method might partially imply that the bioactivity of TGF1 was maintained. To assess the chondrogenic phenotype, the expression of Collagen II within these constructs was examined. The expression of Collagen II has been considered to be sensitive metabolic marker to investigate at the phenotype level, as has been described for chondrocytes isolated from hyaline cartilage tissue. Therefore, in this study, the expression of Collagen II was evaluated as the marker of the phenotype of differentiation of ASCs into chondrocytes. Western blot suggested that the expression of Collagen II was also obviously increased in ASCs-the hybrid MS-TGF when compared to the expression in ASCs-the gelatin MS-TGF at day 14. Such study has provided evidence that chitosan significantly promotes the differentiation of ASCs into chondrocytes. The main reason for this is that chitosan can increase the activity of GFs and could promote the rapid release of TGF-1, which enhances the chondrogenic matrix [22]. In addition, chitosan shares some characteristics with various GAGs and hyaluronic acid present in articular cartilage due to structural resemblance [23], so the hybrid MS around the cells mimics a more natural microenvironment for the ASCs to enhance the production of collagen II. The present in vitro results showed the usefulness of the hybrid MS-TGF as a biomaterial for cultures of ASCs and for differentiation of ASCs into chondrocytes. Take advantage of ASCs-the hybrid MS-TGF, we performed a preliminary study on articular cartilage defects implanted with ASCs-containing hybrid MS in vivo. Our studies suggested that 12 weeks postoperatively both ASCsthe gelatin MS-TGF group and ASCs-the hybrid MS-TGF group stained positive for toluidine blue, which is specific for highly sulfated proteogylcans (the hyaline cartilage phenotype). In addition, histological observations of cartilage repair suggested that ASCsthe hybrid MS-TGF group resulted in better surface zone repair, subcondral bone connection, deeper zone remodeling, as well as the integration of donor with host adjacent cartilage than other two
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groups. We speculated that ASCs seeded onto the hybrid MS should have better differentiation, which promoted the regeneration of cartilage tissue. Conflicts of interest The authors have no conflicts of interest to declare. References [1] Amrami KK, Askari KS, Pagnano MW, et al. Radiologic case study. Abrasion chondroplasty mimicking avascular necrosis. Orthopedics 2002;1018:1107–18. [2] Muller B, Kohn D. Indication for and performance of articular cartilage drilling using the Pridie method. Orthopade 1999;28:4–10. [3] Steadman JR, Rodkey WG, Rodrigo JJ. Microfracture: surgical technique and rehabilitation to treat chondral defects. Clin Orthop Relat Res 2001:S362–9 [Suppl.]. [4] Ringe J, Kaps C, Schmitt B, et al. Porcine mesenchymal stem cells. Induction of distinct mesenchymal cell lineages. Cell Tissue Res 2002;307:321–7. [5] Jankowski RJ, Deasy BM, Huard J. Muscle-derived stem cells. Gene Ther 2002;9:642–7. [6] Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 2001;7:211–28. [7] Safford KM, Hicok KC, Safford SD, et al. Neurogenic differentiation of murine and human adipose-derived stromal cells. Biochem Biophys Res Commun 2002;294:371–9. [8] DeFail AJ, Chu CR, Izzo N, et al. Controlled release of bioactive TGF-beta 1 from microspheres embedded within biodegradable hydrogels. Biomaterials 2006;271:579–1585. [9] Fan H, Hu Y, Li X, et al. Ectopic cartilage formation induced by mesenchymal stem cells on porous gelatin-chondroitin-hyaluronate scaffold containing microspheres loaded with TGF-beta1. Int J Artif Organs 2006;29:602–11. [10] Fan H, Liu H, Zhu R, et al. Comparison of chondral defects repair with in vitro and in vivo differentiated mesenchymal stem cells. Cell Transplant 2007;16:823–32. [11] Suh JK, Matthew HW. Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering: a review. Biomaterials 2000;21:2589–98 [21]. [12] Huang Y, Onyeri S, Siewe M, et al. In vitro characterization of chitosan-gelatin scaffolds for tissue engineering. Biomaterials 2005;26 [7616-7127]. [13] Kim SE, Park JH, Cho YW, et al. Porous chitosan scaffold containing microspheres loaded with transforming growth factor-beta1: implications for cartilage tissue engineering. J Control Release 2003;91:365–74. [14] Wei Y, Hu Y, Lv R, et al. Regulation of adipose-derived adult stem cells differentiating into chondrocytes with the use of rhBMP-2. Cytotherapy 2006;8:570–9. [15] Wakitani S, Goto T, Pineda SJ, et al. Mesenchymal cell-based repair of large, fullthickness defects of articular cartilage. J Bone Joint Surg Am 1994;76:579–92. [16] Pineda S, Pollack A, Stevenson S, et al. A semiquantitative scale for histologic grading of articular cartilage repair. Acta Anat (Basel) 1992;143:335–40. [17] Yoo JU, Barthel TS, Nishimura K, et al. The chondrogenic potential of human bone-marrow-derived mesenchymal progenitor cells. J Bone Joint Surg Am 1998;80:1745–57. [18] Johnstone B, Hering TM, Caplan AI, et al. In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res 1998;238:265–72. [19] Wei Y, Hu Y, Hao W. A morphological study of chondrogenesis by adiposederived adult stem cells induced by recombinant human bone morphogenetic protein 2. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi 2006;20:845–8. [20] Ochiya T, Nagahara S, Sano A, et al. Biomaterials for gene delivery: atelocollagen-mediated controlled release of molecular medicines. Curr Gene Ther 2001;1:31–52. [21] Bigi A, Panzavolta S, Rubini K. Relationship between triple-helix content and mechanical properties of gelatin films. Biomaterials 2004;25:5675–80. [22] Lee JE, Kim KE, Kwon IC, et al. Effects of the controlled-released TGF-beta 1 from chitosan microspheres on chondrocytes cultured in a collagen/chitosan/glycosaminoglycan scaffold. Biomaterials 2004;25:4163–73. [23] Suh JK, Matthew HW. Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering: a review. Biomaterials 2000;21:2589–98.
Original article
Cartilage regeneration using adipose-derived stem cells and the controlled-released hybrid microspheres Yisheng Han ∗ , Yiyong Wei , Shuseng Wang , Yang Song Department of orthopaedics, Xijing Hospital, The Fourth Military Medical University, West Road Changle, Xi’an, China
a r t i c l e
i n f o
Article history: Accepted 19 May 2009 Keywords: Adipose-derived stem cells Gelatin Chitosan Microspheres Cartilage regeneration
a b s t r a c t Objective: This study was to evaluate the effect of hybrid microspheres (MS) composed of gelatin transforming growth factor-beta (TGF-1)-loaded MS and chitosan MS on the enhancement of differentiation of adipose-derived stem cells (ASCs) into chondrocytes in pellet culture in vitro and the reparative capacity of pellet from ASCs and the hybrid MS-TGF used to repair cartilage defects in vivo. Methods: The morphology of the controlled-released MS was observed with scanning electron microscopy (SEM) and mechanical property was also tested in this study. In vitro TGF-1 release was evaluated by an enzyme-linked immunosorbent assay. The protein expression of Collagen II was tested by Western blot. In addition, a preliminary study on cartilage regeneration was also performed in vivo. Results: When chondrogenic differentiation of ASCs in both MS was evaluated, the protein expression of Collagen II became significantly increased for the hybrid MS-TGF, as compared with the gelatin MS-TGF. Mechanical result showed that the hybrid MS was superior to the gelatin MS. Observation of histology in vivo demonstrated that the pellet from ASCs and the hybrid MS-TGF promoted cartilage regeneration in the defects of articular cartilage much better than other groups. Conclusion: Our study demonstrated that the pellet from ASCs and the hybrid MS-TGF can provide an easy and effective way to construct the tissue engineered cartilage in vitro and in vivo. © 2009 Published by Elsevier Masson SAS on behalf of the Société Française de Rhumatologie.
1. Introduction As an avascular tissue with a very slow turnover at the cellular and molecular levels, articular cartilage has a limited capacity for self-repair when damaged as a result of trauma or degenerative diseases. Consequently, the requirement for new cartilage tissue to repair the damaged cartilage is a major concern. Over the past several decades, many solutions have been developed to restore the normal function of the injured cartilage including abrasion, drilling and microfracture [1–3]. Although all of these approaches have proved to be beneficial, none are believed to be optimal for repairing cartilage lesions. Cartilage tissue engineering provides a promising option for the repair of severe damage. The major cell source used to generate engineered cartilage tissue is stem cells. To date, adult stem cells have been identified in various tissues including bone marrow [4], muscle [5] and adipose tissue [6]. Studies have indicated that adult stem cells from adipose tissue contain multipotent progenitor cells, which have the potential to differentiate into osteogenic, chondrogenic, myogenic and neurogenic cells [6,7]. During in vitro expansion, adipose-derived stem cells (ASCs) can maintain a stable undifferentiated status, and
∗ Corresponding author. E-mail address: [email protected] (Y. Han).
pluripotency does not decrease in a growing body. In addition, because adipose tissue is obtained by extraction from the patient in a less invasive manner and provides a large quantity of autologous cells, ASCs represent a fascinating cell source for regenerative medicine. It has been reported that various kinds of growth factors (GFs) affect chondrocyte metabolism and chondrogenesis. Among GFs of potential interest is transforming growth factor-beta (TGF-). As the predominant isoform of TGF-, TGF-1 plays an important role in promoting chondrogenic differentiation in chondroblasts and in mesenchymal cells [8]. Although studies have demonstrated that TGF-1 added directly to the cell culture media induces chondrogenic differentiation of stem cells in vitro, this method decreased the efficacy of TGF-1. Moreover, a suitable concentration was not maintained over an extended time period. So the controlled-release of TGF-1 from a scaffold has become a topic of interest. Gelatin is a partial derivative of collagen. Studies have demonstrated that gelatin MS loaded with TGF-1 could promote chondrocyte growth and differentiation and could maintain the chondrogenic phenotype [9,10]. Moreover, the gelatin MS loaded with TGF-1 could retard the dedifferentiation of chondrocytes when the composite of cells and gelatin is implanted into the body [10]. However, the functional outcome of gelatin is often limited by its inherent poor mechanical stability. In order to increase chondrogenesis and the quality of tissue repair, the use of hybrid
1297-319X/$ – see front matter © 2009 Published by Elsevier Masson SAS on behalf of the Société Française de Rhumatologie. doi:10.1016/j.jbspin.2009.05.013
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microspheres (MS) based on gelatin has recently drawn more attention. Chitosan is a natural polymer, which is structurally similar to various glycosaminoglycans (GAGs) found in articular cartilage and has been reported to be non-toxic, bio-absorbable and to promote would healing [11]. In addition, the study also demonstrated that the mechanical properties of gelatin blended with chitosan were significantly increased when compared to that of gelatin alone, which will promote cartilage regeneration [12]. In this study, we hypothesized that the hybrid TGF1-loaded MS, composed of the gelatin TGF1-loaded MS in combination with the chitosan MS, could provide superior biological effect on the differentiation of ASCs into chondrocytes in pellet culture in comparison with the gelatin MS loaded with TGF-1. To test this hypothesis, the controlled-released hybrid MS were fabricated, and then the mixture of ASCs and the controlled-released hybrid MS was centrifuged to form a pellet. Cell differentiation in the pellet of ASCs-the hybrid MS-TGF was determined and compared with these characteristics in the pellet of ASCs-the gelatin MS-TGF in vitro. In addition, the study is also to evaluate the reparative capacity of the pellet from ASCs and the hybrid MS-TGF used to repair cartilage defects in vivo.
2. Methods 2.1. Preparation of the gelatin MS-TGF and the hybrid MS-TGF The gelatin MS were prepared according to the reported method [9]. Briefly, 1.1 g of acidic gelatin (Sigma Co., St Louis, MO) was dissolved in 7 mL of double-distilled water (dd H2 O) at 53 ◦ C. The aqueous gelatin solution was added dropwise to 30 mL of olive oil and 0.2 mL of Tween 80 (Sigma Co.) while stirring at 500 rpm. The temperature of emulsion was then decreased to 0 ◦ C with constant stirring. After 12 min, 10 mL of chilled acetone (4 ◦ C) was further added. Then, the MS collected were washed with acetone to remove residual oil and cross-linked with 10 mM glutaraldehyde (GA) (Sigma Co.) at 4 ◦ C for 24 h. The cross-linked MS were washed with dd H2 O and counted using a hemocytometer. Then, 500 L of suspension containing 2 × 104 MS was placed in a 1.0 mL microcentrifuge tube for lyophilization. The dried MS (2 × 104 ) were loaded with 50 ng TGF-1 (Protec) by swelling in 5 L of TGF-1 solution (10 ng/L) at pH 7.4. The solution volume is below the MS’ theoretical equilibrium swelling volume to allow for complete TGF-1 absorption. The mixture was gently stirred with a pipette tip and then incubated at 4 ◦ C overnight for thorough swelling, followed by lyophilization. Chitosan MS were prepared according to the reported method [13]. Briefly, chitosan (120 mg) was dissolved in 2% aqueous acetic acid solution (4 ml) until the solution was transparent. The solution was added slowly to HCO-60 solution (5 w/v% in n-octanol) and emulsified at 13,500 rpm by using a homogenizer. After 20 min, 10 ml of TPP solution (10 w/v% in distilled water) was further added dropwise to stabilize the chitosan MS through the electrostatic interaction with TPP. The MS, precipitated in the mixture solvent, were repeatedly washed with excess amounts of isopropyl alcohol and water, and counted using a hemocytometer. Then, 500 L of suspension containing 2 × 104 MS were placed in a 1.0 mL microcentrifuge tube for lyophilization. To construct the hybrid MS-TGF, the gelatin MS-TGF and the chitosan MS (1:1) were crosslinked by 0.3% EDAC at 37 ◦ C for 1 h while being gently stirred. The hybrid MS-TGF was repeatedly washed with phosphate buffered saline (PBS) and then incubated at 4 ◦ C overnight by lyophilization. The morphology of the gelatin MS-TGF and the hybrid MS-TGF was observed with scanning electron microscopy (SEM).
2.2. Mechanical testing measurement Five hundred microlitres of suspension containing MS (the gelatin MS and hybrid MS respectively) were placed in the cylindrical mold for lyophilization, and then gelatin was added into the mold (200 L) for lyophilization to form the cylinder (height 3 mm and diameter 3 mm). Scaffolds were neutralized with alcohol and washed with PBS (pH = 7.4) for 30 min prior to testing (Instron 5569). Tests were performed in 37 ◦ C PBS at a rate of 5 mm/min. 2.3. In vitro TGF-ˇ1 release study In vitro, the gelatin MS-TGF and the hybrid MS-TGF (2 × 104 ) were dispersed into 5 mL of PBS and placed in a shaking water bath at 37 ◦ C, 135 rpm. After 0.5, 1, 3, 5, 7, 10, 14, 21 and 28 days, the suspension was centrifuged at 13,500 rpm to collect the supernatant for TGF-1 analysis. The PBS buffer was added afterward to keep the volume constant. The analysis was performed by an enzyme-linked immunosorbent assay kit (R&D Systems). 2.4. Isolation of adipose-derived stem cells and culture ASCs were isolated from the cervical adipose tissue of adult rabbits (age ≤ 4 months), using a method reported previously [14]. In brief, the tissue obtained was washed with equal volumes of PBS to remove red cells. The adipose tissue was minced finely using surgical scissors and the extracellular matrix was digested for 1 h at 37 ◦ C with 0.15% collagenase (type I; Sigma, St Louis, MO, USA) in PBS. Once digested, enzyme activity was neutralized with culture medium containing DMEM (Gibco, Paisley, UK), 10% FBS (Sijiqing Biological Engineering Materials Co., Hangzhou, China), penicillin (100 U/mL) and streptomycin (100 g/mL). The samples were filtered through a 500 m mesh filter to remove tissue debris. The cell suspension was centrifuged at 800 g to obtain a pellet and the pellet was resuspended in culture medium. The cells were seeded in 25-cm flasks (Corning-Costar, Acton, MA, USA) at density of 4 × 105 cells/cm3 and incubated at 37 ◦ C/5%CO2 . The medium was exchanged after 24 h and then three times a week. At a confluence of more than 80%, cells were detached with 0.25% trypsin and 0.1% EDTA. The cells were passaged five times prior to use. 2.5. Pellet culture Pellet culture was used according to a method reported previously [14]. Briefly, the mixture of ASCs (2 × 105 ) with the gelatin MS-TGF or the hybrid MS-TGF (2 × 104 ) was centrifuged at 500 g for 10 min in 15 mL polypropylene falcon tubes to form a cell pellet. Then, the pellets were cultured in 2 mL basal medium containing 1% FBS, 1 × insulin-transferrin-selenium (ITS) and antibiotics at 37 ◦ C, 5% CO2 , over a period of 2 weeks. The culture medium was changed every other day. 2.6. Histological analysis of pellet Samples were fixed for histological analysis with 4% paraformaldehyde in PBS, embedded in paraffin and sectioned using standard histochemical techniques. Slide sections were stained with H&E. 2.7. Western blot of Collagen II from pellet Protein extracts from chondrocytes, the composites of ASCs-the gelatin MS-TGF and the composites of ASCs-the hybrid MS-TGF were used for Western blot analysis. The protein concentration of the extracts was assessed using a Bradford assay kit. The optical density was measured at 595 nm using a spectrophotometer.
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The protein concentration was interpolated from a standard curve obtained from a serial dilution of bovine serum albumin at a concentration of 0.8 mg/ml. The cell extracts, normalized for total protein content, were resolved using polyacrylamide gel electrophoresis and electrophoretically transferred to a supported nitrocellulose membrane (Amersham, USA). Afterwards, the membranes were blocked in 5% non-fat milk tris-buffered saline tween (TBST) for 2 h, and incubated with primary antibodies, mouse monoclonal anti-collagen II (1:200, Chemicon, USA) at 4 ◦ C overnight. Membranes were then washed three times for 10 min each with TBST, followed by the addition of peroxidase-linked anti-mouse IgG (1:500) and enhanced chemiluminescence (ECL) visualization of the bands. 2.8. Repair of articular defects A cylindrical, full-thickness defect, thickness: 3 mm; diameter: 4 mm, was created using a hand drill with a 4-mm drill bit, penetrating the articular cartilage in the patellar groove of the distal femur in 18 rabbits (the ASCs from them had been prepared as mentioned above). The MS (the gelatin MS-TGF and the hybrid MSTGF, 1 × 105 ) containing ASCs (1 × 106 ) were centrifuged at 500 g for 10 min in the polypropylene falcon tubes to form cell pellets. The pellets were cultured at 37 ◦ C/5%CO2 for 24 h before transplantation experiment. The rabbits were operated on by injection of 2 mL/kg Sumianxin (Agricultural University, Changchun, China) into the muscle. The rabbits were classified into the following three groups: (i) the cartilage defect group (no insertion group), (ii) ASCs-the gelatin MS-TGF group, and (iii) ASCs-the hybrid MSTGF. All rabbits were allowed to move freely after surgery without plaster immobilization. The committee on animal experimentation of Fourth Military Medical University approved all experiments, which met the National Institute of Healthy (NIH) guidelines for the care and use of laboratory animals. Samples were harvested 12 weeks postoperatively. The distal parts of femurs were removed and fixed in 10% paraformaldehyde, decalcified in 10% EDTA and embedded in paraffin, sectioned at 6 m in thickness and stained with toluidine blue. Cartilage repair of the defects (six sections taken from each defect) was evaluated microsopically and scored according to a histological grading scale modified after Wakitani et al. [15] and Pineda et al. [16], consisting of five categories with a total score ranging from 0–20 points. 2.9. Statistical analysis All the data were expressed as the mean ± standard deviation (S.D.). The values in vitro study were analyzed using ANOVA-test. In vivo study histological scores among groups were analyzed with Mann-Whitney test and the Bonferroni method (n = 6). P-value difference less than 0.05 was considered to be statistically significant.
Fig. 1. Scanning electron microscopic images of the gelatin MS-TGF (A) and the hybrid MS-TGF (B) at the original magnification × 4000.
3.3. In vitro TGF-ˇ1 release study A parallel study was undertaken to examine the release kinetics of TGF-1 from the gelatin MS-TGF and the hybrid MS-TGF over 28 days. TGF-1 was released in a biphasic fashion characterized by a burst release at the initial 24 h, which continued until 72 h, after which a plateau was reached. The plateau was maintained until the end of the release period (4 weeks). A fast release on the first day was significantly higher (P < 0.05) for the hybrid MS-TGF (45.2%) when compared to the gelatin MS-TGF (35.9%). Then, the release increased steadily in both the gelatin MS-TGF and the hybrid MSTGF. By day 28, the cumulative release from the hybrid MS-TGF was 92% and it significantly exceeded (P < 0.05) the release from the gelatin MS (77%) (Fig. 3). 3.4. Histological analysis Histological examination using H&E staining indicated that a distinct cartilage-specific morphological appearance and structural characteristics such as lacunae are normally observed in both constructs (Fig. 4). In addition, an unequal distribution of the differentiated ASCs was evident and the differences of cell number were visible between the gelatin MS-TGF and the hybrid MS-TGF. 3.5. Western blot analysis for collagen II Collagen II is the most important component of extracellular matrix (ECM) in native articular cartilage, so its expression in the pellet of ASCs-the MS was tested to evaluate the differentiated status of ASCs. At day 14, the expression of Collagen II in ASCsthe hybrid MS-TGF was significantly higher than that in ASCs-the gelatin MS-TGF (P < 0.05) (Fig. 5). 3.6. In vivo histological evaluation Histological study (Fig. 6) showed that in ASCs-the hybrid MSTGF group, the defects were completely filled with reparative tissue
3. Results 3.1. Characterization of MS-TGF SEM images showed the morphology of the gelatin MS-TGF and the hybrid MS-TGF. Two kinds of MS-TGF were all spherical and had smooth surface (Fig. 1). 3.2. Mechanical properties of the gelatin microspheres (MS) and the hybrid MS Stress-strain curve showed that the stress value was obviously higher in the hybrid MS compared to the gelatin MS when the strain value was the same (Fig. 2).
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Fig. 2. Stress-strain curve of the gelatin microspheres (MS) and the hybrid MS.
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Fig. 6. Histological observation of cartilage repair at postoperative 12 weeks. A. The cartilage defect group. B. Adipose-derived stem cells (ASCs)-the gelatin MS-TGF group. C. ASCs-the hybrid MS-TGF group (toluidine blue staining) (× 100).
Fig. 3. Release profiles of TGF-1 from the gelatin MS-TGF and the hybrid MS-TGF. Data are expressed as mean ± standard deviation (n = 5). The statistical evaluation of the data is detailed in the text.
and the surface of reparative tissue was regular, and integration of the newly formed cartilage with host cartilage was the best among these groups. In addition, extensively metachromatic staining was showed. In ASCs-the gelatin MS-TGF group, the surface of reparative tissue was regular and the integration of donor with host adjacent cartilage was not very good, and the intensity of safranin O declined. The defects were filled with fibrous tissue in the cartilage defect group. The means in Wakitani’s score of the cell-containing MS group exhibited significantly better cartilage regeneration than those of the cartilage defect group at 12 weeks (Table 1). Compared with ASCs-the gelatin MS-TGF group, ASCs-the hybrid MS-TGF group showed much better regeneration of cartilage tissue. 4. Discussion
Fig. 4. H&E staining of adipose-derived stem cells (ASCs)-the gelatin MS-TGF (A × 400) and ASCs-the hybrid MS-TGF (B × 400) at day 14.
Fig. 5. The protein expression of Collagen II for adipose-derived stem cells (ASCs)the gelatin MS-TGF and ASCs-the hybrid MS-TGF at day 14. Data are expressed as mean ± standard deviation (n = 5). *statistically significant relative to ASCs-the gelatin MS-TGF (P < 0.05).
Pellet culture as a culture model results in the formation of three-dimensional structures and mimics precartilage condensations during embryonic development, cell-to-cell interactions among chondrocytes are known to be important in preventing dedifferentiation. Some researchers demonstrated that stem cells plated at high density could accelerate chondrogenic differentiation in vitro [17,18]. Our study had also demonstrated that pellet culture was an appropriate way to control chondrogenesis of ASCs [19]. To further enhance chondrogensis, a modified pellet was formed using ASCs and hybrid MS with controlled-released TGF1. In this study we selected two types of natural polymers to construct the hybrid MS. Gelatin was chosen as the base for the hybrid MS in this study for the following reasons: first, it contains an Arg-Gly-Asp (RGD)-like sequence that promotes cell adhesion and migration; second, it has low immunogenicity and low cost; finally, it can be degraded entirely in vivo and its degradation products are non-toxic [20]. In addition to the characteristics of gelatin mentioned above, it can also provide the release of GFs in a controlled fashion, which can serve to increase the in-growth and biosynthetic ability of cells. However, the use of gelatin for cartilage defect repair has limitations due to its inherent poor mechanical stability [21]. Mechanical property is a key factor in cartilage tissue engineering because the scaffolds should keep proper strength until the newly-formed tissue becomes mechanically competent after implantation. The result of our study showed that the mechanical property of the
Table 1 Results of histological grading (mean values ± standard deviation). n
Control ASCs-the gelatin MS-TGF ASCs-the hybrid MS-TGF
Scores on histologic grading scale
6 6 6
Cell morphology and matrix staining (0–8)
Surface regularity
Integration
Filling of defects
Total
(0–4)
Subchondral bone reconstruction (0–3)
(0–3)
(0–2)
7.8 ± 0.5 5.6 ± 0.3 4.8 ± 0.6
2.9 ± 0.3 1.9 ± 0.4 1.3 ± 0.4
1.8 ± 0.4 1.2 ± 0.4 0.9 ± 0.5
3.8 ± 0.2 1.5 ± 0.1 1.1 ± 0.6
2.7 ± 0.5 1.1 ± 0.1 0.8 ± 0.3
18.2 ± 0.7 9.8 ± 0.5# 7.5 ± 0.4*#
*P < 0.05 compared with ASCs-the gelatin MS-TGF group, #P < 0.05 compared with the cartilage defect group. a The scale has five categories assigning a total score ranging from 0 (best) to 20 (worst).
(0–20)a
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hybrid MS was superior to that of the gelatin MS by the stressstrain curve. Based on the significant increase of the mechanical properties of gelatin blended with chitosan, hybrid MS made from gelatin containing TGF-1 and chitosan were fabricated. The in vitro release of TGF-1 was examined in this study. The study illustrated that there were relatively high release levels of TGF-1 from the hybrid MS when compared to the gelatin MS during the observed period. A possible mechanism is that the positive charge was increased by adding the chitosan MS to the gelatin MS-TGF-1, which resulted in the rapid release of TGF-1 due to electrostatic repulsion. The rapid release will increase the bioactive effect of TGF-1 on stem cells by influencing cell migration, proliferation and differentiation. We also demonstrated that the release profile of TGF-1 in both constructs showed a biphasic model with an initial burst followed by a plateau, which indicated an extended time course for complete release. The earlier burst release is likely caused by the rapid diffusion of TGF-1 located close to the surface of MS, as water uptake plays an important role in modulating the release of TGF-1 in the initial stage. Although the biological activity of TGF-1 was not quantified in this study, the chondrogenic differentiation of ASCs suggested that its activity was preserved. The ELISA method might partially imply that the bioactivity of TGF1 was maintained. To assess the chondrogenic phenotype, the expression of Collagen II within these constructs was examined. The expression of Collagen II has been considered to be sensitive metabolic marker to investigate at the phenotype level, as has been described for chondrocytes isolated from hyaline cartilage tissue. Therefore, in this study, the expression of Collagen II was evaluated as the marker of the phenotype of differentiation of ASCs into chondrocytes. Western blot suggested that the expression of Collagen II was also obviously increased in ASCs-the hybrid MS-TGF when compared to the expression in ASCs-the gelatin MS-TGF at day 14. Such study has provided evidence that chitosan significantly promotes the differentiation of ASCs into chondrocytes. The main reason for this is that chitosan can increase the activity of GFs and could promote the rapid release of TGF-1, which enhances the chondrogenic matrix [22]. In addition, chitosan shares some characteristics with various GAGs and hyaluronic acid present in articular cartilage due to structural resemblance [23], so the hybrid MS around the cells mimics a more natural microenvironment for the ASCs to enhance the production of collagen II. The present in vitro results showed the usefulness of the hybrid MS-TGF as a biomaterial for cultures of ASCs and for differentiation of ASCs into chondrocytes. Take advantage of ASCs-the hybrid MS-TGF, we performed a preliminary study on articular cartilage defects implanted with ASCs-containing hybrid MS in vivo. Our studies suggested that 12 weeks postoperatively both ASCsthe gelatin MS-TGF group and ASCs-the hybrid MS-TGF group stained positive for toluidine blue, which is specific for highly sulfated proteogylcans (the hyaline cartilage phenotype). In addition, histological observations of cartilage repair suggested that ASCsthe hybrid MS-TGF group resulted in better surface zone repair, subcondral bone connection, deeper zone remodeling, as well as the integration of donor with host adjacent cartilage than other two
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groups. We speculated that ASCs seeded onto the hybrid MS should have better differentiation, which promoted the regeneration of cartilage tissue. Conflicts of interest The authors have no conflicts of interest to declare. References [1] Amrami KK, Askari KS, Pagnano MW, et al. Radiologic case study. Abrasion chondroplasty mimicking avascular necrosis. Orthopedics 2002;1018:1107–18. [2] Muller B, Kohn D. Indication for and performance of articular cartilage drilling using the Pridie method. Orthopade 1999;28:4–10. [3] Steadman JR, Rodkey WG, Rodrigo JJ. Microfracture: surgical technique and rehabilitation to treat chondral defects. Clin Orthop Relat Res 2001:S362–9 [Suppl.]. [4] Ringe J, Kaps C, Schmitt B, et al. Porcine mesenchymal stem cells. Induction of distinct mesenchymal cell lineages. Cell Tissue Res 2002;307:321–7. [5] Jankowski RJ, Deasy BM, Huard J. Muscle-derived stem cells. Gene Ther 2002;9:642–7. [6] Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 2001;7:211–28. [7] Safford KM, Hicok KC, Safford SD, et al. Neurogenic differentiation of murine and human adipose-derived stromal cells. Biochem Biophys Res Commun 2002;294:371–9. [8] DeFail AJ, Chu CR, Izzo N, et al. Controlled release of bioactive TGF-beta 1 from microspheres embedded within biodegradable hydrogels. Biomaterials 2006;271:579–1585. [9] Fan H, Hu Y, Li X, et al. Ectopic cartilage formation induced by mesenchymal stem cells on porous gelatin-chondroitin-hyaluronate scaffold containing microspheres loaded with TGF-beta1. Int J Artif Organs 2006;29:602–11. [10] Fan H, Liu H, Zhu R, et al. Comparison of chondral defects repair with in vitro and in vivo differentiated mesenchymal stem cells. Cell Transplant 2007;16:823–32. [11] Suh JK, Matthew HW. Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering: a review. Biomaterials 2000;21:2589–98 [21]. [12] Huang Y, Onyeri S, Siewe M, et al. In vitro characterization of chitosan-gelatin scaffolds for tissue engineering. Biomaterials 2005;26 [7616-7127]. [13] Kim SE, Park JH, Cho YW, et al. Porous chitosan scaffold containing microspheres loaded with transforming growth factor-beta1: implications for cartilage tissue engineering. J Control Release 2003;91:365–74. [14] Wei Y, Hu Y, Lv R, et al. Regulation of adipose-derived adult stem cells differentiating into chondrocytes with the use of rhBMP-2. Cytotherapy 2006;8:570–9. [15] Wakitani S, Goto T, Pineda SJ, et al. Mesenchymal cell-based repair of large, fullthickness defects of articular cartilage. J Bone Joint Surg Am 1994;76:579–92. [16] Pineda S, Pollack A, Stevenson S, et al. A semiquantitative scale for histologic grading of articular cartilage repair. Acta Anat (Basel) 1992;143:335–40. [17] Yoo JU, Barthel TS, Nishimura K, et al. The chondrogenic potential of human bone-marrow-derived mesenchymal progenitor cells. J Bone Joint Surg Am 1998;80:1745–57. [18] Johnstone B, Hering TM, Caplan AI, et al. In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res 1998;238:265–72. [19] Wei Y, Hu Y, Hao W. A morphological study of chondrogenesis by adiposederived adult stem cells induced by recombinant human bone morphogenetic protein 2. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi 2006;20:845–8. [20] Ochiya T, Nagahara S, Sano A, et al. Biomaterials for gene delivery: atelocollagen-mediated controlled release of molecular medicines. Curr Gene Ther 2001;1:31–52. [21] Bigi A, Panzavolta S, Rubini K. Relationship between triple-helix content and mechanical properties of gelatin films. Biomaterials 2004;25:5675–80. [22] Lee JE, Kim KE, Kwon IC, et al. Effects of the controlled-released TGF-beta 1 from chitosan microspheres on chondrocytes cultured in a collagen/chitosan/glycosaminoglycan scaffold. Biomaterials 2004;25:4163–73. [23] Suh JK, Matthew HW. Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering: a review. Biomaterials 2000;21:2589–98.