Hyaluronan production and chondrogenic properties of primary human chondrocyte on gelatin based hematostatic spongostan scaffold
© Klangjorhor et al.; licensee BioMed Central Ltd. 2012
Received: 1 October 2011
Accepted: 5 December 2012
Published: 19 December 2012
Autologous chondrocyte transplantation is a promising technique for treatment of cartilage defects. Three dimensional chondrocyte cultures on a scaffold are widely used to retain the chondrogenic phenotype. Using a biodegradable gelatin scaffold is one option for the cell delivery system, but molecular and histological studies of the method have not yet been done.
We evaluated the chondrogenic property of the primary human chondrocyte on a gelatin scaffold as compared to a collagen scaffold over a period of 21 days. We examined the production of glycosaminoglycan by quantitative and histological analysis. Gene expression of cartilage-associated molecules was assessed by quantitative RT-PCR.
The gelatin scaffold showed the ability to promote chondrocyte expansion, chondrogenic phenotype retention at molecular and mRNA levels.
This scaffold is thus suitable for use as an in vitro model for chondrocyte 3D culture.
KeywordsHuman articular chondrocyte 3D culture Hyaluronan Collagen Gelatin Spongostan Scaffold
Articular chondrocytes have limited ability to self-repair, and usually dedifferentiate to fibroblasts when grown in a monolayer culture. A three–dimensional (3D) culture system can be used to retain the chondrogenic phenotype. Matrix induced autologous chondrocyte implantation (MACI), a third generation ACI technique, was introduced in 1998 and developed as a treatment for chondral and subchondral defects of joint surfaces[1, 2]. During the 5 years following its introduction, the technique underwent clinical improvements and proved suitable for treatment of cartilage defects. A biodegradable scaffold is needed as a vehicle to deliver the precultured chondrocytes to the cartilage defects. The biomaterial scaffolds provide a suitable environment for chondrocytes to maintain their chondrogenic phenotype. Various types of natural substance scaffolds include fibrin, agarose, alginate, collagen[5, 6], chitosan and hyaluronan. In addition supplementary methods include conditional use of 3D culture, hormones, growth factors, variation of oxygen tension, mechanical stimulation and retrovirally transduced SOX9 genes in the chondrocytes. The ideal scaffold should promote the chondrocytes to produce extracellular matrix, which constitutes the regenerated cartilage. It has previously been shown that a gelatin-based scaffold (SpongostanTM, Johnson & Johnson, Norderstedt, Germany) may be used as a 3D chondrocyte matrix. Most studies have reported the total extracellular matrix production rather than glycosaminoglycan concentration. In this study we determined whether human articular chondrocytes embedded in a gelatin based scaffold could retain the chondrogenic properties required for chondrocyte transplantation, and compared this to collagen based scaffolds. We then performed quantitative biochemical and histomorphological analysis over a period of 21 days.
Materials and methods
Cell culture and reagents
Human chondrocytes from non-osteoarthritis joints were harvested from notchpalsty operation from articular knee cartilage of 18–45 years old patients and chondrocytes were isolated and purified by standard protocol. All patients gave consent and all procedures of this study were approved by the Ethical Committee of Chiang Mai University (approval no. 070CT111016). The gelatin scaffold was obtained from Johnson & Johnson (Germany). The collagen scaffold was purchased from BD Bioscience.
All materials and reagents used in this study were purchased from Sigma (Sigma –Aldrich USA) unless stated otherwise.
Cell seeding and culture of scaffold
The primary human chondrocytes were grown and expanded as a monolayer in 10% (v/v) FCS DMEM (Gibco, Germany) containing 10 U/ml penicillin and 100 μg/ml streptomycin up to passage 4 (P4) for use in these experiments. Scaffold cultures were performed by directly seeding the chondrocytes (5×105 cells suspension in 30 μl culture medium), onto the scaffold and leaved the chondrocytes suspension adsorbed and embedded in scaffold for 4 hrs. After that added chondrogenic media compose of DMEM supplemented with 10% FBS, 1X ITS +1, 25 μg/ml ascorbic acid 2-phosphate, 10-7 M dexamethasone, 10 ng/ml TGF-β and 100 ng/ml IGF-1 and allowed to adhere in a humidified incubator (37°C, 5% CO2) for 24 hrs. The chondrocytes embedded in the scaffolds were transferred to new culture wells. The numbers of non-adhering cells were measured by trypsinization of the culture well adhering cells and counted by a hemacytometer.
Extracellular matrix (ECM) biomolecules assay
The sulfated glycosaminoglycan (s-GAG) was determined using the dimethylmethylene blue (DMMB) assay with chondroitin 6-sulfate (CS-C: 0–60 μg/ml) as a standard. The DMMB solution was added to the sample and standards prior to reading absorbance values at 525 nm in a microplate reader spectrophotometer.
Hyaluronic acid was measured using a competitive based ELISA as previously described. Briefly, samples or standard HA (Healon®) at various concentrations (19–10,000 ng/ml) in PBS pH 7.4, were added to 1.5 ml plastic tubes containing biotinylated HABPs (1:200 in 0.05 M Tris–HCl buffer, pH 8.6). The tubes were incubated at room temperature for 1 hr. and then samples were added to the microplate, which was pre-coated with umbilical cord HA (100 μl/well of 10 mg/ml), and blocked with 1% BSA (150 μl/well). The plate was then incubated at room temperature for 1 hr. The wells were subsequently washed and 100 μl of peroxidase-conjugated anti-biotin antibody (1:2,000 dilution in PBS) were added. The plate was incubated at room temperature for another hour. The reaction was stopped after 10 minutes with 50 μl/well of 4 M sulfuric acid and the absorbance was determined using a microplate reader at 492/690 nm. The concentration of HA in samples was calculated by reference to a standard curve.
The total amount of DNA was measured by the Hoecht 33258 fluorescent dye assay. A 20 μl aliquot was diluted in 2 ml of dye/buffer solution and the fluorescence of the samples was evaluated for excitation at 450 nm and emission at 555 nm by a spectrophotometer. A standard curve was prepared from known concentrations of herring sperm.
RNA extraction and gene expression analysis
Total RNA was isolated from scaffold culture using an RNA extraction kit (Amershame Bioscience) following the manufacturer’s protocol and the reagents in all extraction steps are RNases free solution. Total RNA (1 μg) was converted to cDNA using a RevertAid TM First Strand cDNA synthesis kit (MBI Fermentas, Germany). For determination of gene expression, SYBR Green detection was used and the values were normalized using glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Real-time quantitative polymerase chain reaction (PCR) was performed in a DNA Engine (ABi 7500) using SYBR GREENER qPCR UNIVERSAL (Invitrogen, USA). Primer sequences (from Invitrogen) are as follows:
Aggrecan core protein Forward 5’-3’ CTGTTCAGGGACAGAATGTGCT; Reverse 5’-3’ TCGATATGCTTCACAGTTCTAGGG: Collagen type I Forward 5’-3’ TTTTGGCCATCTCTTCCTTCA; Reverse 5’-3’ TGTGGATGCCTCTTGGGTATC: Collagen type II Forward 5’-3’ TCCTCTTCTTGAGCTGGACTCATTCT; Reverse 5’-3’ CGCTCTGCAAACTGGAGGTC: SOX9 Forward 5’-3’ GAAGGTGAAGGTCGGAGTC; Reverse 5’-3’ GAAGATGGTGATGGGATTTC. GAPDH Forward 5’-3’ GAAGGTGAAGGTCGGAGTC; Reverse 5’-3’ GAAGATGGTGATGGGATTTC. Relative expression levels for each primer set were normalized to the expression of GAPDH by the 2 -ΔCT method.
For light microscopy, the samples were fixed in a 0.1 M phosphate-buffer (pH7.4) solution of 1% (v/v) paraformaldehyde and 1.25% (v/v) glutaraldehyde, dehydrated in alcohol and embedded in polymethylmethacrylate. Sections (4 μm) were stained with haematoxylin-eosin (H&E) and Safranin O.
For scanning electron microscopy, the samples were prefixed for 24 hrs. at 4°C with 2% (v/v) glutaraldehyde and 2% (v/v) tannic acid in 0.1 M sodium-cacodylate buffer (pH 7.4). Matrices were dehydrated through a graded series of ethanol and acetone; critical point dried using a CPD 7501 critical point Drier, mounted on stubs and spattered with an ultrathin layer of gold in a SEM coating system using SPI-MODULETM Spatter Coater. Specimens were studied using a JEOL 5910 LV SEM apparatus at an accelerating voltage of 15 kV.
Data shown are representative of repeated experiments. Triplicate samples were used for chemical and biochemical assay. Duplicate samples were used for microscopy analysis. The data are expressed as mean ± SD. The significance of differences between groups was tested using non-parametric Wilcoxan rank-sum tests. Statistical significance was considered as p < 0.05.
Production of glycosaminoglycan molecules
The chondrogenic mRNA profile
Scanning electron microscopy
Gelatin, a porous form of denatured collagen, is used as a potential cell delivery system and to support bovine chondrocyte metabolism. It has been used as a scaffold for cartilage tissue engineering[20, 21]. In addition, gelatin scaffold (SpongostanTM Johnson & Johnson) has suitable properties as a 3D chondrocyte matrix for bovine chondrocytes. A collagen scaffold, with its inherent cartilage biological matrix, has been demonstrated to be a cell supporter and to enhance chondrogenic culturing of chondrocytes[12, 22–24]. Based on the fact that the biological composition of gelatin is derived from collagen, a collagen scaffold should match its physio-biochemical properties, in vivo biodegradable property, and on the evidence from chondrocyte culture, we selected collagen scaffolds for comparison. We evaluated gelatin scaffolds (SpongostanTM Johnson & Johnson) as chondrocyte 3D cultures for in vitro hyaline cartilage regeneration using human articular chondrocytes. The biochemical and histological analyses were designed to examine the influence of gelatin and collagen scaffolds on chondrocyte ECM synthesis over a 21-day culture period. Initially, we counted the numbers of non-adhering chondrocytes for estimate amount of chondrocytes which adhered into the scaffold to evaluate adhesive activity of chondrocyte to the scaffold and we found the level of chondrocytes adhering to the gelatin scaffold was significant less than in the collagen scaffold, due to the larger pore size (100–300 μm vs 100–200 μm). Gelatin is derived from denatured collagen, which retains an RGD motif peptide for cell adhesion and penetration. Even during initial cell adhering, there was evidence that chondrocyte seeded gelatin scaffolds had higher cell proliferation and chondrocyte matrix production, as shown by the DNA content and wet weight of the chondrocyte seeded gelatin scaffold being significantly higher than for the collagen scaffold after 21 days of culture.
The sulfated glycosaminoglycan production was not significantly different for both releasing and matrix forms. This was similar to the results from s-GAG staining among the two types of scaffold, which had no significant difference in matrix Safranin O stain intensity. Interestingly, the human chondrocytes seeded on the gelatin scaffold exhibited the higher HA production, for both matrix component and released form, than cells seeded on the collagen scaffold. The HA can serve as the core molecule for HA binding domains and linking proteins of cartilage matrix proteoglycans. Moreover, via binding to cell surface receptors, this molecule plays a role in cell proliferation, morphogenesis and wound repair.
Our study examined quantitative chondrogenic gene expression at 7 day of a 21 days culture. At 7 days cultivation, the chondrocytes seeded collagen scaffold expressed higher levels of hyaline cartilage specific genes (Aggrecan core protein, Collagen type II and SOX9) than the gelatin scaffold. At day 14, a decrease in transcription factor SOX9 gene expression was observed. This protein is crucial for induction and regulation of the chondrocyte phenotype. Aggrecan and Collagen type II expression are regulated by this transcription factor. A decrease in SOX9 induction affects the Aggrecan and Collagen type II expression. Thus, higher SOX9 expression on day 21 of the chondrocytes seeded gelatin scaffold potentially promoted the higher levels of Aggrecan and Collagen type II gene expression compared to the chondrocyte seeded collagen scaffold.
Our study described for the first time the preparation and characterization of primary human chondrocytes cultured in absorbable haemostatic gelatin (SpongostanTM) scaffold in extracellular matrix production including HA and GAG and specific chondrocyte differentiation gene markers. This observation indicated that the gelatin scaffold retained the chondrogenic response including the cells had not de-differentiation to the fibroblasts, enhanced extracellular matrix (HA and GAG) production and more potential to stabilized the expressions or mRNA levels of chondrogenic differentiation gene markers including SOX9 transcription factor, Aggrecan core protein and Collagen type II mRNA in late period of cultivation. Moreover in Collagen type I, dedifferentiation marker level was lower than chondrocytes cultured in collagen scaffold suggested that gelatin scaffold reduced de-differention of the cells. The phenotype maintaining ability of chondrocytes seeded on a scaffold is important for repairing cartilage defects. To our knowledge, this is the first study to report the quantification of HA production of a chondrocyte seeded scaffold. The higher HA production of the chondrocyte seeded gelatin scaffold over 21 days promoted cell proliferation and matrix synthesis and expression of cartilage specific matrix proteins.
In conclusion, our investigation has shown that a gelatin scaffold is adequate to support chondrocytes, and to induce extracellular matrix production of both HA and s-GAG. The high production of matrix HA from the chondrocyte seeded gelatin scaffold may prevent dedifferentiation via transmembrane receptor CD 44, resulting in promotion of cell proliferation and enhanced matrix molecule production. In addition, this scaffold supported expression of many cartilage matrix genes, including SOX9, Aggrecan and Collagen type II. Since a gelatin scaffold is a cheaper medical device, and showed the ability to support chondrocyte expansion and retention of the chondrogenic phenotype, it should be suitable as an alternative for use in in vitro 3D human chondrocyte culture as a regenerative cartilage model.
The National Research Council of Thailand (Research Program of Drug, Chemical, Medical Material and Equipment to PK) The Royal Golden Jubilee Ph.D. Program, (Grant No. PHD/0148/2551 to JK), The Graduate School Chiang Mai University (to JK), Faculty of Medicine Research Fund, Chiang Mai University (to DP and PN) and CMU are jointly funded this work. We thank Dr. Dale E. Taneyhill for proofreading the manuscript.
- Behrens P, Bitter T, Kurz B, Russlies M: Matrix-associated autologous chondrocyte transplantation/implantation (MACT/MACI)-5-year follow-up. Knee. 2006, 13: 194-202. 10.1016/j.knee.2006.02.012.View ArticlePubMedGoogle Scholar
- Behrens P, Ehlers EM, Kochermann KU, Rohwedel J, Russlies M, Plotz W: [New therapy procedure for localized cartilage defects. Encouraging results with autologous chondrocyte implantation]. MMW Fortschr Med. 1999, 141: 49-51.PubMedGoogle Scholar
- Rahfoth B, Weisser J, Sternkopf F, Aigner T, von der Mark K, Brauer R: Transplantation of allograft chondrocytes embedded in agarose gel into cartilage defects of rabbits. Osteoarthr Cartil. 1998, 6: 50-65. 10.1053/joca.1997.0092.View ArticlePubMedGoogle Scholar
- Hauselmann HJ, Masuda K, Hunziker EB, Neidhart M, Mok SS, Michel BA, Thonar EJ: Adult human chondrocytes cultured in alginate form a matrix similar to native human articular cartilage. Am J Physiol. 1996, 271: C742-752.PubMedGoogle Scholar
- Fuss M, Ehlers EM, Russlies M, Rohwedel J, Behrens P: Characteristics of human chondrocytes, osteoblasts and fibroblasts seeded onto a type I/III collagen sponge under different culture conditions. A light, scanning and transmission electron microscopy study. Ann Anat. 2000, 182: 303-310. 10.1016/S0940-9602(00)80002-3.View ArticlePubMedGoogle Scholar
- Ronziere MC, Roche S, Gouttenoire J, Demarteau O, Herbage D, Freyria AM: Ascorbate modulation of bovine chondrocyte growth, matrix protein gene expression and synthesis in three-dimensional collagen sponges. Biomaterials. 2003, 24: 851-861. 10.1016/S0142-9612(02)00418-0.View ArticlePubMedGoogle Scholar
- Chenite A, Chaput C, Wang D, Combes C, Buschmann MD, Hoemann CD, Leroux JC, Atkinson BL, Binette F, Selmani A: Novel injectable neutral solutions of chitosan form biodegradable gels in situ. Biomaterials. 2000, 21: 2155-2161. 10.1016/S0142-9612(00)00116-2.View ArticlePubMedGoogle Scholar
- Grigolo B, Roseti L, Fiorini M, Fini M, Giavaresi G, Aldini NN, Giardino R, Facchini A: Transplantation of chondrocytes seeded on a hyaluronan derivative (hyaff-11) into cartilage defects in rabbits. Biomaterials. 2001, 22: 2417-2424. 10.1016/S0142-9612(00)00429-4.View ArticlePubMedGoogle Scholar
- Tsukazaki T, Usa T, Matsumoto T, Enomoto H, Ohtsuru A, Namba H, Iwasaki K, Yamashita S: Effect of transforming growth factor-beta on the insulin-like growth factor-I autocrine/paracrine axis in cultured rat articular chondrocytes. Exp Cell Res. 1994, 215: 9-16. 10.1006/excr.1994.1307.View ArticlePubMedGoogle Scholar
- Jakob M, Demarteau O, Schafer D, Hintermann B, Dick W, Heberer M, Martin I: Specific growth factors during the expansion and redifferentiation of adult human articular chondrocytes enhance chondrogenesis and cartilaginous tissue formation in vitro. J Cell Biochem. 2001, 81: 368-377. 10.1002/1097-4644(20010501)81:2<368::AID-JCB1051>3.0.CO;2-J.View ArticlePubMedGoogle Scholar
- Domm C, Schunke M, Christesen K, Kurz B: Redifferentiation of dedifferentiated bovine articular chondrocytes in alginate culture under low oxygen tension. Osteoarthr Cartil. 2002, 10: 13-22. 10.1053/joca.2001.0477.View ArticlePubMedGoogle Scholar
- Lee CR, Grodzinsky AJ, Spector M: Biosynthetic response of passaged chondrocytes in a type II collagen scaffold to mechanical compression. J Biomed Mater Res A. 2003, 64: 560-569.View ArticlePubMedGoogle Scholar
- Tew SR, Li Y, Pothacharoen P, Tweats LM, Hawkins RE, Hardingham TE: Retroviral transduction with SOX9 enhances re-expression of the chondrocyte phenotype in passaged osteoarthritic human articular chondrocytes. Osteoarthr Cartil. 2005, 13: 80-89. 10.1016/j.joca.2004.10.011.View ArticlePubMedGoogle Scholar
- Anders JO, Mollenhauer J, Beberhold A, Kinne RW, Venbrocks RA: Gelatin-based haemostyptic spongostan as a possible three-dimensional scaffold for a chondrocyte matrix?: an experimental study with bovine chondrocytes. J Bone Joint Surg Br. 2009, 91: 409-416.View ArticlePubMedGoogle Scholar
- Ratcliffe A, Beauvais PJ, Saed-Nejad F, Shurety W, Caterson B: Synovial fluid analyses detect and differentiate proteoglycan metabolism in canine experimental models of osteoarthritis and disuse atrophy. Agents Actions Suppl. 1993, 39: 63-67.PubMedGoogle Scholar
- Phitak T, Choocheep K, Pothacharoen P, Pompimon W, Premanode B, Kongtawelert P: The effects of p-hydroxycinnamaldehyde from alpinia galanga extracts on human chondrocytes. Phytochemistry. 2009, 70: 237-243. 10.1016/j.phytochem.2008.11.010.View ArticlePubMedGoogle Scholar
- Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(−delta delta C(T)) method. Methods. 2001, 25: 402-408. 10.1006/meth.2001.1262.View ArticlePubMedGoogle Scholar
- Nuernberger S, Cyran N, Albrecht C, Redl H, Vecsei V, Marlovits S: The influence of scaffold architecture on chondrocyte distribution and behavior in matrix-associated chondrocyte transplantation grafts. Biomaterials. 2011, 32: 1032-1040. 10.1016/j.biomaterials.2010.08.100.View ArticlePubMedGoogle Scholar
- Goodstone N, Gargiulo B, Cartwright A, Ashton B: Effects of high molecular weight hyaluronan on chondrocytes cultured within spongostan, a resorbable gelatin sponge. Eur Cell Mater. 2002, 4 (Suppl. 2): 9-10.Google Scholar
- Pesakova V, Stol M, Adam M: Comparison of the influence of gelatine and collagen substrates on growth of chondrocytes. Folia Biol (Praha). 1990, 36: 264-270.Google Scholar
- Ponticiello MS, Schinagl RM, Kadiyala S, Barry FP: Gelatin-based resorbable sponge as a carrier matrix for human mesenchymal stem cells in cartilage regeneration therapy. J Biomed Mater Res. 2000, 52: 246-255. 10.1002/1097-4636(200011)52:2<246::AID-JBM2>3.0.CO;2-W.View ArticlePubMedGoogle Scholar
- Buma P, Pieper JS, van Tienen T, van Susante JL, van der Kraan PM, Veerkamp JH, van den Berg WB, Veth RP, van Kuppevelt TH: Cross-linked type I and type II collagenous matrices for the repair of full-thickness articular cartilage defects–a study in rabbits. Biomaterials. 2003, 24: 3255-3263. 10.1016/S0142-9612(03)00143-1.View ArticlePubMedGoogle Scholar
- De Franceschi L, Grigolo B, Roseti L, Facchini A, Fini M, Giavaresi G, Tschon M, Giardino R: Transplantation of chondrocytes seeded on collagen-based scaffold in cartilage defects in rabbits. J Biomed Mater Res A. 2005, 75: 612-622.View ArticlePubMedGoogle Scholar
- Lee CR, Breinan HA, Nehrer S, Spector M: Articular cartilage chondrocytes in type I and type II collagen-GAG matrices exhibit contractile behavior in vitro. Tissue Eng. 2000, 6: 555-565. 10.1089/107632700750022198.View ArticlePubMedGoogle Scholar
- Wang QG, Hughes N, Cartmell SH, Kuiper NJ: The composition of hydrogels for cartilage tissue engineering can influence glycosaminoglycan profile. Eur Cell Mater. 2010, 19: 86-95.PubMedGoogle Scholar
- Turley EA, Noble PW, Bourguignon LY: Signaling properties of hyaluronan receptors. J Biol Chem. 2002, 277: 4589-4592. 10.1074/jbc.R100038200.View ArticlePubMedGoogle Scholar
- de Crombrugghe B, Lefebvre V, Nakashima K: Regulatory mechanisms in the pathways of cartilage and bone formation. Curr Opin Cell Biol. 2001, 13: 721-727. 10.1016/S0955-0674(00)00276-3.View ArticlePubMedGoogle Scholar
- Sekiya I, Tsuji K, Koopman P, Watanabe H, Yamada Y, Shinomiya K, Nifuji A, Noda M: SOX9 Enhances aggrecan gene promoter/enhancer activity and is up-regulated by retinoic acid in a cartilage-derived cell line, TC6. J Biol Chem. 2000, 275: 10738-10744. 10.1074/jbc.275.15.10738.View ArticlePubMedGoogle Scholar
- Li Y, Tew SR, Russell AM, Gonzalez KR, Hardingham TE, Hawkins RE: Transduction of passaged human articular chondrocytes with adenoviral, retroviral, and lentiviral vectors and the effects of enhanced expression of SOX9. Tissue Eng. 2004, 10: 575-584. 10.1089/107632704323061933.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.