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Development of a cartilage composite utilizing porous tantalum, fibrin, and rabbit chondrocytes for treatment of cartilage defect

Development of a cartilage composite utilizing porous tantalum, fibrin, and rabbit chondrocytes... Objective: Functional tissue engineering has emerged as a potential means for treatment of cartilage defect. Development of a stable cartilage composite is considered to be a good option. The aim of the study was to observe whether the incorporation of cultured chondrocytes on porous tantalum utilizing fibrin as a cell carrier would promote cartilage tissue formation. Methods: Rabbit articular chondrocytes were cultured and seeded onto tantalum with fibrin as temporary matrix in a composite, which was divided into three groups. The first group was kept in vitro while a total of 12 constructs were implanted into the dorsum of mice for the second and third groups. The implanted tissues were harvested after 4 weeks (second group) and after 8 weeks (third group). Specific characteristic of cartilage growth were studied by histological and biochemical assessment, immunohistochemistry, and quantitative PCR analysis. Results: Histological and biochemical evaluation of the formed cartilage using hematoxylin and eosin and Alcian blue staining showed lacunae chondrocytes embedded in the proteoglycan rich matrix. Dimethylmethylene blue assay demonstrated high glycosaminoglycans content in the removed tissue following 8 weeks of implantation. Immunohistochemistry results showed the composites after implantation expressed high collagen type II. Quantitative PCR results confirmed a significant increase in cartilage associated genes expression (collagen type II, AggC, Sox 9) after implantation. Conclusion: Tantalum scaffold with fibrin as cell carrier promotes chondrocyte proliferation and cartilaginous tissue formation. Producing hyaline cartilage within a stable construct of tantalum and fibrin has a potential for treatment of cartilage defect. Keywords: Cartilage composite, Porous tantalum, Chondrocyte proliferation, Cartilage defect, Fibrin Introduction ineffective response to injury, articular cartilage has limited Around two million individuals are affected worldwide potential to heal, even so in larger defects. Partial thickness with arthritis [1]. These individuals require hospitalization, injuries do not heal and merely stimulate minimal reaction which is a matter of concern. Total joint replacement is to adjacent chondrocytes in the form of cell replication and the usual end-stage treatment, but implant longevity is an matrix turnover, whereas full thickness injuries penetrating issue as more than half of the affected individuals is below subchondral bone produce normal healing response but 65 years old [2-7]. Damage to articular cartilage leads to eventually fills the defect with fibrocartilage [9]. Fibrocarti- premature arthritis. This chondral lesion was reported to lage resists to tension, in contrast to normal hyaline cartil- be present in 60% of all patients aged between 40–50 years age which resists compressive forces. Therefore, there is an during arthroscopy [1,7,8]. Due to its avascular nature and urgent need for a method for effective biological healing and regeneration of cartilage to prevent premature arthritis. * Correspondence: drkortho@gmail.com There are various methods of operative treatment Department of Orthopaedic and Traumatology, Faculty of Medicine, employed to treat osteochondral injury. Marrow stimula- Universiti Kebangsaan Malaysia, Jalan Yaacob Latiff, Bandar Tun Razak, tion and resurfacing techniques have been advocated but 56000 Cheras, Kuala Lumpur, Malaysia Full list of author information is available at the end of the article © 2015 Jamil et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Jamil et al. Journal of Orthopaedic Surgery and Research (2015) 10:27 Page 2 of 9 they have their limitations while dealing with large de- Wire Cut (Hitachi, H-Cut 203 M20) in thickness of fects and producing hyaline cartilage [9-15]. Autologous 0.5 mm and Milling Machine Cut to make holes Ø1.0 chondrocyte implantation (ACI) which was brought into on the both surface of tantalum (Figure 1). attention by Brittberg et al. in 1994 is quite promising but this technique is expensive and highly technically Chondrocyte cell preparation dependent [10]. It requires two surgeries and needs la- Six New Zealand white rabbits age 2 months old and boratory support to grow the cells [13]. weighting 2.0–2.3 kilograms were used as experimental Functional tissue engineering is a novel approach to animals. Blood was taken to get autologous plasma and enhance tissue regeneration and provides the possibility serum before the animal was sacrificed with overdosage of producing tissue that is biomechanically, biochemically, of phenobarbital. Full-thickness cartilage was harvested and histomorphologically similar to the native tissues [7]. from the knee, hip joints, and patella articular surface Scientists and engineers working in this promising field under aseptic technique. Each cartilage was separated are taking steps to make those ideas a reality, working to from the perichondrium and subchondrium bone, supply biological substitutes or living tissue. A previous minced into small pieces (1 mm ), washed with Dul- study by Munirah et al. for articular cartilage restoration becco phosphate buffer saline (DPBS; pH 7.2; Gibco, focused on engineering autologous cartilage construct Grand Island, NY, USA) containing 100 μ/ml penicillin using human and ovine chondrocytes incorporated with and 100 μ/ml streptomycin (PBS, Gibco). Finally, the the autologous fibrin as biomaterial [16]. The pre-clinical samples were washed with DPBS one more time. Carti- study, conducted in sheep, was designed to evaluate the lages were digested using 0.3% collagenase type II (Gibco) performance of the autologous ‘chondrocytes-fibrin’ con- in a shaker incubator (Stuart Scientific, Redhill, UK) at 37°C struct implantation in a simulated clinical application for 90 min. Samples were centrifuged (500 × g)for 10 min prior to undertaking the definitive pre-clinical and clinical to get the cell pellet. The supernatant was removed and the investigations. However, it has its own limitation. Resulted cell pellet was washed with 15 ml DPBS to remove the ‘chondrocytes-fibrin’ constructs were too soft to hold into remaining enzyme. The sample was centrifuged for 10 min the defect independently. Technically, periosteal patch to get the final pellet cells. Isolated chondrocytes were were used to secure the implant into the defect. There is continuous quest for the development of a new and more reliable technique to restore or recon- struct osteochondral defects. The development of a composite with the biochemical and mechanical proper- ties of an osteochondral graft with better integration properties and without the need for autologous osteo- chondral graft harvesting or allogeneic tissue would be attractive. Tantalum, an elemental metal which is strong, biocompatible, and corrosive-resistant, has been widely used in the field of orthopedics due to its low modulus of elasticity and low frictional characteristics. It also has high, interconnected porosity with excellent bone in growth properties. In the present study, we incorporated cultured chondrocytes on porous tantalum utilizing fi- brin as a cell carrier to support in vitro chondrogenesis. Following this, we compared it with constructs im- planted for 4 and 8 weeks, which hypothetically has su- perior cartilaginous development. Materials and methods The study was conducted after receiving the ethical ap- proval from the animal and research ethics committee of our university. The project approval number from Universiti Kebangsaan Malaysia was FF-307-2009. Tantalum preparation Figure 1 Tantalum scaffold in 10 mm in diameter and 0.5 mm All scaffolds were prepared from 2 cm in diameter and in thickness. 10 cm length of Zimmer tantalum screw using Machine Jamil et al. Journal of Orthopaedic Surgery and Research (2015) 10:27 Page 3 of 9 seeded in six well plates containing the culture medium Histological and biochemical assessment (Ham’s F12 and Dulbecco’s modified medium 1:1 + 1% as- The obtained cartilage underwent histology and bio- corbic acid + 10% autologous serum + antibiotic/antimyco- chemical evaluation. Constructs were fixed in 10% for- tic + 1% glutamine). All cultures were maintained in 5% malin for 24 h at 4°C and processed using standard CO incubator (Jouan, Duguay, Trouin, SH) at 37°C with histological technique which finally embedded in paraf- every 3 days medium change. Once confluence, the primary fin. The tissues were cross sectioned (5 μm thick) and cultures were trypsinized using trypsin-EDTA 0.125% stained with hematoxylin and eosin, and Alcian blue ac- (Gibco). The cells pellet were then cultured in large-scale cording to standard procedures. For biochemical assess- 175 cm culture flask (Falkon, Franklin Lake, NJ, USA) at a ment, the amount of glucosaminoglycans (GAGs) was density of 5,000 cells/cm . Chondrocyte morphologic fea- measured using the dimethylmethylene blue (DMMB) tureswereexaminedevery dayusing inverted lightmicro- assay (12) and the total amount of GAGs per dry weight scope (Olympus, Shinjukuku, Tokyo). Upon confluence, the of formed cartilage (μg/ml/mg) was recorded. cells were trypsinized and mix with plasma before put on to the tantalum. Fibrin polymerization took place after CaCl GAG quantification solution was added into the mixture. The cell-fibrin- For quantitative measuring of GAGs, the total GAGs tantalum constructs were maintained for 3 days in the cul- content of cell/polymer constructs was determined using ture before implantation. It was cultured in the same papain digestion and the dimethylmethylene blue dye medium for cell expansion, in the six-well dishes and main- method. Samples were lyophilized for 24 h and then tained in the CO incubator. digested under sterile conditions with papain type III (Sigma) at 125 mg/ml in a buffer of 0.1 M NaH PO , 2 4 5 mM methylenediaminetetraacetic acid, and 5 mM Construct implantation cysteine hydrochloride at pH 7.0 overnight at 60°C be- Under aseptic technique, surgery was performed under fore the dye was added. Dimethylmethylene blue stock sedation. Cocktail drug which consist of ketamine, xyla- solution was made using dimethylmethylene blue, sodium zine, and zoletil was given according to body weight chloride, glycine, sodium aziade in 1 N hydrochloride, and intramuscular. Surgical incision was made at the dorsum water. A spectrophotometer set at a wavelength of 520 nm of nude mice, and two constructs were implanted on the was used to measure the optical density of the digested left and right side of the dorsum (Figure 2). The skin samples. Glycosaminoglycan was measured and reported was then sutured using 6/0 vicryl (12 complexes totally in micrograms per milliliter per milligram of dry weight at six mice). Care of the nude mice was carried out follow- tissue. ing the animal facility guideline of the Animal Unit, UKM. Immunohistochemistry Formalin-fixed tissues were sectioned and treated with Harvest of composite proteinase K at 37°C for 60 min before washed three Three mice were sacrificed after 4 weeks and another times with tris buffered saline (TBS, DAKO Cytoma- three mice were sacrificed after 8 weeks to remove the tion). The sections were then treated with peroxidase constructs. block at 37°C for 10 min prior to incubation with anti- body. Two antibodies were used, anti-type I and anti- type II collagen were diluted 1:150 with diluent (DAKO Cytomation) and applied to different sections for 40 min at 37°C. After washing with TBS, the sections were reacted with horseradish peroxidase (HRP) for 40 min at 37°C. After washing again with TBS, the signal was fi- nally visualized as a brown reaction product from the peroxidase substrate 3,3′-diaminobenzidine (DAB). Quantitative gene expression by real-time PCR Total RNA was extracted from removed tissue engineered using TRI reagent (Molecular Research Center, Cincinnati) according to the manufacturer. Cartilage differentiation (type II collagen, SOX9 transcription factor, and aggrecan core protein), dedifferentiation (type I collagen), and hyper- trophy (type X collagen) gene expression level was quanti- Figure 2 Construct implanted over dorsum of mice. fied by real-time PCR technique. Primer sequence used was Jamil et al. Journal of Orthopaedic Surgery and Research (2015) 10:27 Page 4 of 9 designed with Primer 3 software based on the GeneBank and felt to be firm, resisting to compression resembling a database sequences corresponding to the specific gene Ac- normal hyaline cartilage (Figure 4). cession Number. The reaction kinetic and specificity of each primer set was verified with standard curve and melt- Histological results ing profile. The quantitative RT-PCR protocol was per- The histological results at day 1 in vitro and 4 weeks formed in a Bio-Rad iCycler with profile of cDNA synthesis and 8 weeks in vivo constructs were variable. In vitro for 30 min at 50°C, pre-denaturation for 2 min at 94°C and samples demonstrated immature lacunae cells with PCR amplification for 38 cycles with 10 s at 94°C, 10 s at scanty basophilic matrix background. In vivo samples at 60°C, and 30 s at 72°C. This series of cycles was followed by 4 weeks implantation had more evenly distributed lacu- melt curve analysis to check the reaction specificity. The nae cells, but immature with reactive nucleus in the tis- data was analyzed using Bio-Rad iCycler software. The ex- sue. Samples at 8 weeks implantation revealed evenly pression level of each targeted gene was normalized to the distributed lacunae embedded within a basophilic matrix house keeping gene - GAPDH. (Figure 5). At both 4 and 8 weeks, the tantalum remains intact and incorporated as a composite with the cells Statistical analysis and fibrin. Data for GAGs amount in each construct at all three envi- Alcian blue staining as specific staining for proteogly- ronments (in vitro, in vivo, 4 and 8 weeks) were collected can showed immature cells with scanty hyalinized matrix from 16 samples. Values were presented as mean ± stand- background in in vitro samples. Following 4 weeks im- ard error of mean (SEM). ANOVA and Student’s t test plantation, the cells appeared rounded in clusters with were used to compare data between groups. Differences extracellular matrix-stained blue, indicating production were considered significant when p < 0.05. Data collected of proteoglycan. In vivo samples at 8 weeks demonstrated from quantitative parameter was analyzed using independ- abundant mature cells in clusters, with extensive staining ent t test or Mann–Whitney test. Values were presented throughout the extracellular matrix (Figure 6). as mean ± SEM. Differences at 5% level were considered significant. All analyses were performed using SPSS 10.0 GAG quantification of constructs software. A mean amount of GAGs per dry weight of formed tis- sue in in vitro, 4 weeks in vivo, and 8 weeks in vivo was Results 47.32 ± 3.12, 58.37 ± 1.28, and 72.40 ± 2.72, respectively Cellular morphology and construct gross appearance (Figure 7). Statistical analysis using ANOVA method In monolayer culture, chondrocytes exhibited small and showed increasing amount of GAGs between groups polygonal shape and they continued to proliferate and from in vitro (day 1) to in vivo (4 and 8 weeks) which is reached confluency after 1 week (Figure 3). After con- significant (p < 0.005). This demonstrated high-quality struct implantation, all nude mice survived without any cartilage tissue formed with increasing implantation period clinical signs of morbidity or mortality. Grossly, the con- in the in vivo environment. structs demonstrated stable form of implant with no signs Quantitative comparison between in vitro and 4 weeks of tissue reaction. After 4 and 8 weeks implantation, three in vivo construct using Student t test demonstrated sig- nude mice were sacrificed at 4 weeks and another three nificant difference between the total amount of pro- nude mice at 8 weeks to harvest the constructs. Tissue- duced GAGs (p = 0.013). Comparison of total amount of engineered cartilage appeared white, smooth, glistening, GAGs per dry weight between 4 and 8 weeks in vivo Figure 3 Chondrocytes morphology at primary culture. (A) Small, polygonal shape at early stage. (B) Chondrocytes reached confluency after 1 week in culture. Magnification × 200. Jamil et al. Journal of Orthopaedic Surgery and Research (2015) 10:27 Page 5 of 9 and less lacunar formed. We observed moderate expression towards collagen type I around the underdeveloped pericel- lular matrix but no expression for collagen type II (Figure 8). In vivo construct showed strong expression towards collagen type II with brown discoloration pericellular and throughout the matrix. Weak immunopositivity was observed towards collagen type I (Figure 9). Real-time PCR analysis on the constructs Cartilage-associated genes (collagen type II, aggrecan core protein, Sox 9 transcription factor) showed signifi- cantly higher expression in in vivo constructs at 4 and 8 weeks compared to in vitro construct. This proved the greater ability of in vivo tissue-engineered cartilage to produce mature cartilage phenotype. However, we observed Figure 4 Construct of tantalum-chondrocyte-fibrin after no significant difference between in vivo constructs at 4 implantation. and 8 weeks, except for aggrecan core protein (Figure 10). Fibrocartilage (collagen type I) and hypertrophic (col- demonstrated significant difference as well (p = 0.006). lagen type X) markers showed moderately high gene ex- Samples at 8 weeks implantation formed more high-quality pression levels. There was significant difference between cartilage with higher amount of GAGs production. in vitro and in vivo constructs at 4 and 8 weeks but no difference was observed between in vivo constructs at Immunohistochemistry of constructs the same period (Figure 11). Following implantation, tissue-engineered cartilage showed progression from immature tissues towards maturity by 4 Discussion and 8 weeks. By 8 weeks, the histological feature exhibited Areas of research regarding cartilage repair have essentially lacunar being formed with increase in extracellular matrix. focusedonculturedcells supported in the engineered tissue. In vitro construct exhibited poorly distributed cartilage cells The delivery of this tissue is one of the challenges which Figure 5 H&E staining of tissue in in vitro (A), 4 weeks in vivo (B), and 8 weeks in vivo (C). In vitro construct showed immature cells with scanty basophilic background, while both in vivo constructs demonstrated evenly distributed lacunae cells within basophilic matrix. However, 8-week construct exhibited more mature cells throughout extracellular matrix. Jamil et al. Journal of Orthopaedic Surgery and Research (2015) 10:27 Page 6 of 9 Figure 6 Alcian blue staining for proteoglycan of tissue in vitro (A), 4 weeks in vivo (B), and 8 weeks in vivo (C). Formed tissue obtained from 8 weeks in vivo construct revealed abundant cells in clusters with positive staining throughout the extracellular matrix compared to 4 weeks in vivo and in vitro construct. include developing an appropriate scaffold and adhesive to to normal rabbit cartilage. This cartilage from femoral con- provide matrix for the cells to grow. The term ‘chondrocon- dyles were made into osteochondral plugs and tested against ductive’ was described by Gordon et al. and defined as pro- the composite. It showed a stress–strain curve with charac- viding a scaffold for the growth of cartilage and supporting teristics typical of normal cartilage responding to a load. structures [17]. They found that porous tantalum is chondro- These findings have been the basis of further studies to con- conductive in vitro in dynamic environment. The potential sider cartilage-tantalum composite as an option for resur- of porous tantalum has been further evaluated by Mardones facing and arthroplasty procedures. The abovementioned et al. in the development of cartilage-tantalum composite technique has limitation in term of making different thick- [18].Inthisstudy,periosteumfromrabbits were placed on ness of cartilage from periosteum tissue. In the present study, top of porous tantalum cylinders and cultured under chon- we used fibrin to bind cultured chondrocytes and seeded drogenic conditions for 6 weeks. The findings show hyaline- onto tantalum in order to increase the feasibility of making like cartilage on the surface of the cylinders while the pores various thickness of cartilage tissue. of the scaffold were filled with fibrous fixation. Mechanical The limitation of the study was the relatively short study testing was also performed which showed properties similar period. An animal study by Shao et al. using allogeneic Figure 7 Comparison of mean of GAGs amount in various environments (μg/ml/mg) in increasing pattern from in vitro to in vivo construct at 4 and 8 weeks. (p < 0.05). Jamil et al. Journal of Orthopaedic Surgery and Research (2015) 10:27 Page 7 of 9 Figure 8 Immunohistochemical staining for in vitro construct shows brownish deposition for localization of collagen type I (A) but no expression for collagen type II (B). bone marrow mesenchymal stem cells (BMSC) seeded spindle-shaped and elongated during monolayer culture. onto fibrin glue matrix and medical-grade polycaprolatone These findings are similar to previous studies, which (mPCL) revealed deterioration of the transplant after portrays the dedifferentiation in culture as the cultured 6 months, despite early good results [19]. Admittedly, if chondrocytes lost their phenotype to adopt fibroblastic the present study could have been conducted over the traits [15,21,22]. Our 2-month results also demonstrated same time period with the previous reported study, we that cultured chondrocytes within the fibrin and tantalum could have compared it better. Furthermore, the subcuta- scaffold are able to differentiate and produce hyaline-like neous environment of our construct on the dorsum of cartilage tissue. Histological evaluation of in vivo samples nude mice might not be representative of a true clinical revealed chondroblast and chondrocytes surrounded by situation where intraarticular environment is involved. matrix containing hyaline. Seeded cell on tantalum in vitro Mrosek et al. reported an in vivo study where cylindrical was uniform and homogenous and contained immature osteochondral defects were created on the medial and lat- chondrocytes with low concentration of GAGs in the hya- eral condyles of ten rabbits and filled with tantalum/perios- linized matrix, while in implanted constructs, the total teum or poly-epsilon-caprolactone/periosteum biosynthetic amount of GAGs per dry weight of tissue significantly in- composites [20]. Even though subchondral bone regener- creased. This was further proven by strong immunopositivity ation was excellent, neo-cartilage formation from periosteum by immunohistochemistry at 4 and 8 weeks, respectively. It supported by a scaffold was inconsistent [20]. However, also means that by 2 months, the cartilage has not featured Munirah et al. proved that autologous chondrocyte-fibrin any age-related changes yet. Studies with longer duration construct (ACFC) promotes early chondrogenesis by indu- need to be done to evaluate the integrity of the cartilage. We cing hyaline-like cartilage regeneration at 12 weeks post- also found that cartilage-associated genes (collagen type II, surgery in a sheep model [16]. Biomechanical testing is also AggC, Sox 9) showed high gene expression after 8 weeks. appropriate as the next step for tantalum-chondrocyte-fibrin This supports our hypothesis and suggested that the cartil- composite. age composite had produced a good matrix for the chon- In the present study, the tissue-engineered cartilage drocytes to differentiate. Similar findings were reported by showed morphological features of polygonal that became previous studies of human articular chondrocytes [15,23]. Figure 9 Immunohistochemical staining of in vivo construct shows strong reaction towards collagen type II (A) but weak reaction with collagen type I with brown discoloration extracellularly (B). Jamil et al. Journal of Orthopaedic Surgery and Research (2015) 10:27 Page 8 of 9 Figure 11 Comparison between in vivo constructs (4 and 8 weeks) to in vitro for the quantitative RT-PCR analysis (p<0.05) of gene expression of collagen type I (A) and collagen type X (B). Native isolated RNA from normal native cartilage. of a single surgery without the use of a periosteal patch which can be unstable. Figure 10 Comparison between in vivo constructs (4 and 8 weeks) to Conclusion in vitro for the quantitative RT-PCR analysis (p<0.05). It determined We showed that tantalum scaffold with fibrin as cell carrier the gene expression of collagen type II (A), aggrecan (B), and Sox 9 (C). promotes cellular proliferation and cartilaginous tissue Native isolated RNA from normal native cartilage. ormation of rabbit articular chondrocytes. Engineered car- tilage resulted from in vivo-implanted construct demon- Fibrocartilage and hypertrophic markers even though strated high-quality hyaline-like tissue by histological and present showed much lower expression level. Sasano et al. biochemical assessment, GAG quantification, immunohis- also discovered that chondrocytes synthesize collagen type tochemistry, and real-time PCR. This early results highlight I and accumulate the protein in the matrix during his the potential of tantalum-chondrocyte-fibrin composite in study of rat tibial articular cartilage [15,24]. treatment of cartilage defect. These findings are in accordance with previous studies Competing interests in proving that autologous chondrocyte and fibrin compos- The authors declare that they have no competing interests. ite produced good-quality cartilage-like tissue [21,25-32]. In Authors’ contributions addition, we overcome the concern of fibrin glue detach- KJ carried out the animal studies, participated in the laboratory assessments, ment and degradation by providing a good scaffold with and drafted the manuscript. SLN carried out the histological and biochemical tantalum. Tantalum usage has been well established in assessments, immunohistochemistry, PCR analysis, and statistical analysis. SJ participated in the cell preparation and harvest of composite. NHY conceived of clinical settings due to its biocompatibility and highly in- the study and participated in the design of the study. KHC conceived of the terconnected porosity which permits physiologic bone study, participated in its design and coordination, and helped to draft the ingrowth and healing [17,33-41]. By 8 weeks, this tantalum- manuscript. All authors read and approved the final manuscript. chondrocyte-fibrin composite show good promise in pro- Acknowledgements viding solution for cartilage defect. Clinically, this cartilage The source of funding for the study was from the Research Fund of construct may be implanted arthroscopically and remain in Universiti Kebangsaan Malaysia. We would like to thank Professor Srijit Das situ with a stable scaffold. This advantage offers the option for his help and contribution in completing the manuscript. Jamil et al. Journal of Orthopaedic Surgery and Research (2015) 10:27 Page 9 of 9 Author details 22. Ishak MF, Chua KH, Asma A, Saim L, Aminuddin BS, Ruszymah BH, et al. Department of Orthopaedic and Traumatology, Faculty of Medicine, Stem cell genes are poorly expressed in chondrocytes from microtic Universiti Kebangsaan Malaysia, Jalan Yaacob Latiff, Bandar Tun Razak, cartilage. Int J Pediatr Otorhinolaryngol. 2011;75:835–40. 56000 Cheras, Kuala Lumpur, Malaysia. Department of Physiology, Faculty of 23. Munirah S, Kim SH, Ruszymah BH, Khang G. The use of fibrin and poly Medicine, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, (lactic-co-glycolic acid) hybrid scaffold for articular cartilage tissue 50300 Kuala Lumpur, Malaysia. engineering: an in vivo analysis. Eur Cell Mater. 2008;15:41–52. 24. Sasano Y, Furusawa M, Ohtani H, Mizoguchi I, Takahashi I, Kagayama M. Received: 11 September 2014 Accepted: 14 January 2015 Chondrocytes synthesize type I collagen and accumulate the protein in the matrix during development of rat tibial articular cartilage. 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J Biomed Mater Res. 2005;75(2):229–33. revision using a porous tantalum biomaterial: a case series. J Arthroplasty. 18. Mardones RM, Reinholz GG, Fitzsimmons JS, Zobitz ME, An KN, Lewallen DG, 2008;24(7):1068–73. et al. Development of a biologic prosthetic composite for cartilage repair. 40. Meneghini RM, Lewallen DG, Hanssen AD. Use of porous tantalum Tissue Eng. 2005;11(9–10):1368–78. metaphyseal cones for severe tibial bone loss during revision total knee 19. Shao XX, Hutmacher DW, Ho ST, Goh JC, Lee EH. Evaluation of a hybrid replacement. Surgical technique. J Bone Joint Surg Am. 2009;91(2):131–8. scaffold/cell construct in repair of high-load-bearing osteochondral defects 41. Patil N, Lee K, Goodman SB. Porous tantalum in hip and knee reconstructive in rabbits. Biomaterials. 2006;27(7):1071–80. surgery. J Biomed Mater Res B Appl Biomater. 2009;89(1):242–51. 20. Mrosek EH, Schagemann JC, Chung HW, Fitzsimmons JS, Yaszemski MJ, Mardones RM, et al. Porous tantalum and poly-epsilon-caprolactone biocomposites for osteochondral defect repair: preliminary studies in rabbits. J Orthop Res. 2010;28(2):141–8. 21. Idrus R, Chua KH, Munirah S, Noruddin N, Aminuddin BS. Tissue engineered cartilage with different human chondrocyte sources: articular, auricular and nasal septum. Med J Islamic World Acad Sci. 2005;15(1):5–12. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Orthopaedic Surgery and Research Springer Journals

Development of a cartilage composite utilizing porous tantalum, fibrin, and rabbit chondrocytes for treatment of cartilage defect

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Copyright © 2015 by Jamil et al.; licensee BioMed Central.
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Medicine & Public Health; Orthopedics; Surgical Orthopedics
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1749-799X
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10.1186/s13018-015-0166-z
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25889942
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Abstract

Objective: Functional tissue engineering has emerged as a potential means for treatment of cartilage defect. Development of a stable cartilage composite is considered to be a good option. The aim of the study was to observe whether the incorporation of cultured chondrocytes on porous tantalum utilizing fibrin as a cell carrier would promote cartilage tissue formation. Methods: Rabbit articular chondrocytes were cultured and seeded onto tantalum with fibrin as temporary matrix in a composite, which was divided into three groups. The first group was kept in vitro while a total of 12 constructs were implanted into the dorsum of mice for the second and third groups. The implanted tissues were harvested after 4 weeks (second group) and after 8 weeks (third group). Specific characteristic of cartilage growth were studied by histological and biochemical assessment, immunohistochemistry, and quantitative PCR analysis. Results: Histological and biochemical evaluation of the formed cartilage using hematoxylin and eosin and Alcian blue staining showed lacunae chondrocytes embedded in the proteoglycan rich matrix. Dimethylmethylene blue assay demonstrated high glycosaminoglycans content in the removed tissue following 8 weeks of implantation. Immunohistochemistry results showed the composites after implantation expressed high collagen type II. Quantitative PCR results confirmed a significant increase in cartilage associated genes expression (collagen type II, AggC, Sox 9) after implantation. Conclusion: Tantalum scaffold with fibrin as cell carrier promotes chondrocyte proliferation and cartilaginous tissue formation. Producing hyaline cartilage within a stable construct of tantalum and fibrin has a potential for treatment of cartilage defect. Keywords: Cartilage composite, Porous tantalum, Chondrocyte proliferation, Cartilage defect, Fibrin Introduction ineffective response to injury, articular cartilage has limited Around two million individuals are affected worldwide potential to heal, even so in larger defects. Partial thickness with arthritis [1]. These individuals require hospitalization, injuries do not heal and merely stimulate minimal reaction which is a matter of concern. Total joint replacement is to adjacent chondrocytes in the form of cell replication and the usual end-stage treatment, but implant longevity is an matrix turnover, whereas full thickness injuries penetrating issue as more than half of the affected individuals is below subchondral bone produce normal healing response but 65 years old [2-7]. Damage to articular cartilage leads to eventually fills the defect with fibrocartilage [9]. Fibrocarti- premature arthritis. This chondral lesion was reported to lage resists to tension, in contrast to normal hyaline cartil- be present in 60% of all patients aged between 40–50 years age which resists compressive forces. Therefore, there is an during arthroscopy [1,7,8]. Due to its avascular nature and urgent need for a method for effective biological healing and regeneration of cartilage to prevent premature arthritis. * Correspondence: drkortho@gmail.com There are various methods of operative treatment Department of Orthopaedic and Traumatology, Faculty of Medicine, employed to treat osteochondral injury. Marrow stimula- Universiti Kebangsaan Malaysia, Jalan Yaacob Latiff, Bandar Tun Razak, tion and resurfacing techniques have been advocated but 56000 Cheras, Kuala Lumpur, Malaysia Full list of author information is available at the end of the article © 2015 Jamil et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Jamil et al. Journal of Orthopaedic Surgery and Research (2015) 10:27 Page 2 of 9 they have their limitations while dealing with large de- Wire Cut (Hitachi, H-Cut 203 M20) in thickness of fects and producing hyaline cartilage [9-15]. Autologous 0.5 mm and Milling Machine Cut to make holes Ø1.0 chondrocyte implantation (ACI) which was brought into on the both surface of tantalum (Figure 1). attention by Brittberg et al. in 1994 is quite promising but this technique is expensive and highly technically Chondrocyte cell preparation dependent [10]. It requires two surgeries and needs la- Six New Zealand white rabbits age 2 months old and boratory support to grow the cells [13]. weighting 2.0–2.3 kilograms were used as experimental Functional tissue engineering is a novel approach to animals. Blood was taken to get autologous plasma and enhance tissue regeneration and provides the possibility serum before the animal was sacrificed with overdosage of producing tissue that is biomechanically, biochemically, of phenobarbital. Full-thickness cartilage was harvested and histomorphologically similar to the native tissues [7]. from the knee, hip joints, and patella articular surface Scientists and engineers working in this promising field under aseptic technique. Each cartilage was separated are taking steps to make those ideas a reality, working to from the perichondrium and subchondrium bone, supply biological substitutes or living tissue. A previous minced into small pieces (1 mm ), washed with Dul- study by Munirah et al. for articular cartilage restoration becco phosphate buffer saline (DPBS; pH 7.2; Gibco, focused on engineering autologous cartilage construct Grand Island, NY, USA) containing 100 μ/ml penicillin using human and ovine chondrocytes incorporated with and 100 μ/ml streptomycin (PBS, Gibco). Finally, the the autologous fibrin as biomaterial [16]. The pre-clinical samples were washed with DPBS one more time. Carti- study, conducted in sheep, was designed to evaluate the lages were digested using 0.3% collagenase type II (Gibco) performance of the autologous ‘chondrocytes-fibrin’ con- in a shaker incubator (Stuart Scientific, Redhill, UK) at 37°C struct implantation in a simulated clinical application for 90 min. Samples were centrifuged (500 × g)for 10 min prior to undertaking the definitive pre-clinical and clinical to get the cell pellet. The supernatant was removed and the investigations. However, it has its own limitation. Resulted cell pellet was washed with 15 ml DPBS to remove the ‘chondrocytes-fibrin’ constructs were too soft to hold into remaining enzyme. The sample was centrifuged for 10 min the defect independently. Technically, periosteal patch to get the final pellet cells. Isolated chondrocytes were were used to secure the implant into the defect. There is continuous quest for the development of a new and more reliable technique to restore or recon- struct osteochondral defects. The development of a composite with the biochemical and mechanical proper- ties of an osteochondral graft with better integration properties and without the need for autologous osteo- chondral graft harvesting or allogeneic tissue would be attractive. Tantalum, an elemental metal which is strong, biocompatible, and corrosive-resistant, has been widely used in the field of orthopedics due to its low modulus of elasticity and low frictional characteristics. It also has high, interconnected porosity with excellent bone in growth properties. In the present study, we incorporated cultured chondrocytes on porous tantalum utilizing fi- brin as a cell carrier to support in vitro chondrogenesis. Following this, we compared it with constructs im- planted for 4 and 8 weeks, which hypothetically has su- perior cartilaginous development. Materials and methods The study was conducted after receiving the ethical ap- proval from the animal and research ethics committee of our university. The project approval number from Universiti Kebangsaan Malaysia was FF-307-2009. Tantalum preparation Figure 1 Tantalum scaffold in 10 mm in diameter and 0.5 mm All scaffolds were prepared from 2 cm in diameter and in thickness. 10 cm length of Zimmer tantalum screw using Machine Jamil et al. Journal of Orthopaedic Surgery and Research (2015) 10:27 Page 3 of 9 seeded in six well plates containing the culture medium Histological and biochemical assessment (Ham’s F12 and Dulbecco’s modified medium 1:1 + 1% as- The obtained cartilage underwent histology and bio- corbic acid + 10% autologous serum + antibiotic/antimyco- chemical evaluation. Constructs were fixed in 10% for- tic + 1% glutamine). All cultures were maintained in 5% malin for 24 h at 4°C and processed using standard CO incubator (Jouan, Duguay, Trouin, SH) at 37°C with histological technique which finally embedded in paraf- every 3 days medium change. Once confluence, the primary fin. The tissues were cross sectioned (5 μm thick) and cultures were trypsinized using trypsin-EDTA 0.125% stained with hematoxylin and eosin, and Alcian blue ac- (Gibco). The cells pellet were then cultured in large-scale cording to standard procedures. For biochemical assess- 175 cm culture flask (Falkon, Franklin Lake, NJ, USA) at a ment, the amount of glucosaminoglycans (GAGs) was density of 5,000 cells/cm . Chondrocyte morphologic fea- measured using the dimethylmethylene blue (DMMB) tureswereexaminedevery dayusing inverted lightmicro- assay (12) and the total amount of GAGs per dry weight scope (Olympus, Shinjukuku, Tokyo). Upon confluence, the of formed cartilage (μg/ml/mg) was recorded. cells were trypsinized and mix with plasma before put on to the tantalum. Fibrin polymerization took place after CaCl GAG quantification solution was added into the mixture. The cell-fibrin- For quantitative measuring of GAGs, the total GAGs tantalum constructs were maintained for 3 days in the cul- content of cell/polymer constructs was determined using ture before implantation. It was cultured in the same papain digestion and the dimethylmethylene blue dye medium for cell expansion, in the six-well dishes and main- method. Samples were lyophilized for 24 h and then tained in the CO incubator. digested under sterile conditions with papain type III (Sigma) at 125 mg/ml in a buffer of 0.1 M NaH PO , 2 4 5 mM methylenediaminetetraacetic acid, and 5 mM Construct implantation cysteine hydrochloride at pH 7.0 overnight at 60°C be- Under aseptic technique, surgery was performed under fore the dye was added. Dimethylmethylene blue stock sedation. Cocktail drug which consist of ketamine, xyla- solution was made using dimethylmethylene blue, sodium zine, and zoletil was given according to body weight chloride, glycine, sodium aziade in 1 N hydrochloride, and intramuscular. Surgical incision was made at the dorsum water. A spectrophotometer set at a wavelength of 520 nm of nude mice, and two constructs were implanted on the was used to measure the optical density of the digested left and right side of the dorsum (Figure 2). The skin samples. Glycosaminoglycan was measured and reported was then sutured using 6/0 vicryl (12 complexes totally in micrograms per milliliter per milligram of dry weight at six mice). Care of the nude mice was carried out follow- tissue. ing the animal facility guideline of the Animal Unit, UKM. Immunohistochemistry Formalin-fixed tissues were sectioned and treated with Harvest of composite proteinase K at 37°C for 60 min before washed three Three mice were sacrificed after 4 weeks and another times with tris buffered saline (TBS, DAKO Cytoma- three mice were sacrificed after 8 weeks to remove the tion). The sections were then treated with peroxidase constructs. block at 37°C for 10 min prior to incubation with anti- body. Two antibodies were used, anti-type I and anti- type II collagen were diluted 1:150 with diluent (DAKO Cytomation) and applied to different sections for 40 min at 37°C. After washing with TBS, the sections were reacted with horseradish peroxidase (HRP) for 40 min at 37°C. After washing again with TBS, the signal was fi- nally visualized as a brown reaction product from the peroxidase substrate 3,3′-diaminobenzidine (DAB). Quantitative gene expression by real-time PCR Total RNA was extracted from removed tissue engineered using TRI reagent (Molecular Research Center, Cincinnati) according to the manufacturer. Cartilage differentiation (type II collagen, SOX9 transcription factor, and aggrecan core protein), dedifferentiation (type I collagen), and hyper- trophy (type X collagen) gene expression level was quanti- Figure 2 Construct implanted over dorsum of mice. fied by real-time PCR technique. Primer sequence used was Jamil et al. Journal of Orthopaedic Surgery and Research (2015) 10:27 Page 4 of 9 designed with Primer 3 software based on the GeneBank and felt to be firm, resisting to compression resembling a database sequences corresponding to the specific gene Ac- normal hyaline cartilage (Figure 4). cession Number. The reaction kinetic and specificity of each primer set was verified with standard curve and melt- Histological results ing profile. The quantitative RT-PCR protocol was per- The histological results at day 1 in vitro and 4 weeks formed in a Bio-Rad iCycler with profile of cDNA synthesis and 8 weeks in vivo constructs were variable. In vitro for 30 min at 50°C, pre-denaturation for 2 min at 94°C and samples demonstrated immature lacunae cells with PCR amplification for 38 cycles with 10 s at 94°C, 10 s at scanty basophilic matrix background. In vivo samples at 60°C, and 30 s at 72°C. This series of cycles was followed by 4 weeks implantation had more evenly distributed lacu- melt curve analysis to check the reaction specificity. The nae cells, but immature with reactive nucleus in the tis- data was analyzed using Bio-Rad iCycler software. The ex- sue. Samples at 8 weeks implantation revealed evenly pression level of each targeted gene was normalized to the distributed lacunae embedded within a basophilic matrix house keeping gene - GAPDH. (Figure 5). At both 4 and 8 weeks, the tantalum remains intact and incorporated as a composite with the cells Statistical analysis and fibrin. Data for GAGs amount in each construct at all three envi- Alcian blue staining as specific staining for proteogly- ronments (in vitro, in vivo, 4 and 8 weeks) were collected can showed immature cells with scanty hyalinized matrix from 16 samples. Values were presented as mean ± stand- background in in vitro samples. Following 4 weeks im- ard error of mean (SEM). ANOVA and Student’s t test plantation, the cells appeared rounded in clusters with were used to compare data between groups. Differences extracellular matrix-stained blue, indicating production were considered significant when p < 0.05. Data collected of proteoglycan. In vivo samples at 8 weeks demonstrated from quantitative parameter was analyzed using independ- abundant mature cells in clusters, with extensive staining ent t test or Mann–Whitney test. Values were presented throughout the extracellular matrix (Figure 6). as mean ± SEM. Differences at 5% level were considered significant. All analyses were performed using SPSS 10.0 GAG quantification of constructs software. A mean amount of GAGs per dry weight of formed tis- sue in in vitro, 4 weeks in vivo, and 8 weeks in vivo was Results 47.32 ± 3.12, 58.37 ± 1.28, and 72.40 ± 2.72, respectively Cellular morphology and construct gross appearance (Figure 7). Statistical analysis using ANOVA method In monolayer culture, chondrocytes exhibited small and showed increasing amount of GAGs between groups polygonal shape and they continued to proliferate and from in vitro (day 1) to in vivo (4 and 8 weeks) which is reached confluency after 1 week (Figure 3). After con- significant (p < 0.005). This demonstrated high-quality struct implantation, all nude mice survived without any cartilage tissue formed with increasing implantation period clinical signs of morbidity or mortality. Grossly, the con- in the in vivo environment. structs demonstrated stable form of implant with no signs Quantitative comparison between in vitro and 4 weeks of tissue reaction. After 4 and 8 weeks implantation, three in vivo construct using Student t test demonstrated sig- nude mice were sacrificed at 4 weeks and another three nificant difference between the total amount of pro- nude mice at 8 weeks to harvest the constructs. Tissue- duced GAGs (p = 0.013). Comparison of total amount of engineered cartilage appeared white, smooth, glistening, GAGs per dry weight between 4 and 8 weeks in vivo Figure 3 Chondrocytes morphology at primary culture. (A) Small, polygonal shape at early stage. (B) Chondrocytes reached confluency after 1 week in culture. Magnification × 200. Jamil et al. Journal of Orthopaedic Surgery and Research (2015) 10:27 Page 5 of 9 and less lacunar formed. We observed moderate expression towards collagen type I around the underdeveloped pericel- lular matrix but no expression for collagen type II (Figure 8). In vivo construct showed strong expression towards collagen type II with brown discoloration pericellular and throughout the matrix. Weak immunopositivity was observed towards collagen type I (Figure 9). Real-time PCR analysis on the constructs Cartilage-associated genes (collagen type II, aggrecan core protein, Sox 9 transcription factor) showed signifi- cantly higher expression in in vivo constructs at 4 and 8 weeks compared to in vitro construct. This proved the greater ability of in vivo tissue-engineered cartilage to produce mature cartilage phenotype. However, we observed Figure 4 Construct of tantalum-chondrocyte-fibrin after no significant difference between in vivo constructs at 4 implantation. and 8 weeks, except for aggrecan core protein (Figure 10). Fibrocartilage (collagen type I) and hypertrophic (col- demonstrated significant difference as well (p = 0.006). lagen type X) markers showed moderately high gene ex- Samples at 8 weeks implantation formed more high-quality pression levels. There was significant difference between cartilage with higher amount of GAGs production. in vitro and in vivo constructs at 4 and 8 weeks but no difference was observed between in vivo constructs at Immunohistochemistry of constructs the same period (Figure 11). Following implantation, tissue-engineered cartilage showed progression from immature tissues towards maturity by 4 Discussion and 8 weeks. By 8 weeks, the histological feature exhibited Areas of research regarding cartilage repair have essentially lacunar being formed with increase in extracellular matrix. focusedonculturedcells supported in the engineered tissue. In vitro construct exhibited poorly distributed cartilage cells The delivery of this tissue is one of the challenges which Figure 5 H&E staining of tissue in in vitro (A), 4 weeks in vivo (B), and 8 weeks in vivo (C). In vitro construct showed immature cells with scanty basophilic background, while both in vivo constructs demonstrated evenly distributed lacunae cells within basophilic matrix. However, 8-week construct exhibited more mature cells throughout extracellular matrix. Jamil et al. Journal of Orthopaedic Surgery and Research (2015) 10:27 Page 6 of 9 Figure 6 Alcian blue staining for proteoglycan of tissue in vitro (A), 4 weeks in vivo (B), and 8 weeks in vivo (C). Formed tissue obtained from 8 weeks in vivo construct revealed abundant cells in clusters with positive staining throughout the extracellular matrix compared to 4 weeks in vivo and in vitro construct. include developing an appropriate scaffold and adhesive to to normal rabbit cartilage. This cartilage from femoral con- provide matrix for the cells to grow. The term ‘chondrocon- dyles were made into osteochondral plugs and tested against ductive’ was described by Gordon et al. and defined as pro- the composite. It showed a stress–strain curve with charac- viding a scaffold for the growth of cartilage and supporting teristics typical of normal cartilage responding to a load. structures [17]. They found that porous tantalum is chondro- These findings have been the basis of further studies to con- conductive in vitro in dynamic environment. The potential sider cartilage-tantalum composite as an option for resur- of porous tantalum has been further evaluated by Mardones facing and arthroplasty procedures. The abovementioned et al. in the development of cartilage-tantalum composite technique has limitation in term of making different thick- [18].Inthisstudy,periosteumfromrabbits were placed on ness of cartilage from periosteum tissue. In the present study, top of porous tantalum cylinders and cultured under chon- we used fibrin to bind cultured chondrocytes and seeded drogenic conditions for 6 weeks. The findings show hyaline- onto tantalum in order to increase the feasibility of making like cartilage on the surface of the cylinders while the pores various thickness of cartilage tissue. of the scaffold were filled with fibrous fixation. Mechanical The limitation of the study was the relatively short study testing was also performed which showed properties similar period. An animal study by Shao et al. using allogeneic Figure 7 Comparison of mean of GAGs amount in various environments (μg/ml/mg) in increasing pattern from in vitro to in vivo construct at 4 and 8 weeks. (p < 0.05). Jamil et al. Journal of Orthopaedic Surgery and Research (2015) 10:27 Page 7 of 9 Figure 8 Immunohistochemical staining for in vitro construct shows brownish deposition for localization of collagen type I (A) but no expression for collagen type II (B). bone marrow mesenchymal stem cells (BMSC) seeded spindle-shaped and elongated during monolayer culture. onto fibrin glue matrix and medical-grade polycaprolatone These findings are similar to previous studies, which (mPCL) revealed deterioration of the transplant after portrays the dedifferentiation in culture as the cultured 6 months, despite early good results [19]. Admittedly, if chondrocytes lost their phenotype to adopt fibroblastic the present study could have been conducted over the traits [15,21,22]. Our 2-month results also demonstrated same time period with the previous reported study, we that cultured chondrocytes within the fibrin and tantalum could have compared it better. Furthermore, the subcuta- scaffold are able to differentiate and produce hyaline-like neous environment of our construct on the dorsum of cartilage tissue. Histological evaluation of in vivo samples nude mice might not be representative of a true clinical revealed chondroblast and chondrocytes surrounded by situation where intraarticular environment is involved. matrix containing hyaline. Seeded cell on tantalum in vitro Mrosek et al. reported an in vivo study where cylindrical was uniform and homogenous and contained immature osteochondral defects were created on the medial and lat- chondrocytes with low concentration of GAGs in the hya- eral condyles of ten rabbits and filled with tantalum/perios- linized matrix, while in implanted constructs, the total teum or poly-epsilon-caprolactone/periosteum biosynthetic amount of GAGs per dry weight of tissue significantly in- composites [20]. Even though subchondral bone regener- creased. This was further proven by strong immunopositivity ation was excellent, neo-cartilage formation from periosteum by immunohistochemistry at 4 and 8 weeks, respectively. It supported by a scaffold was inconsistent [20]. However, also means that by 2 months, the cartilage has not featured Munirah et al. proved that autologous chondrocyte-fibrin any age-related changes yet. Studies with longer duration construct (ACFC) promotes early chondrogenesis by indu- need to be done to evaluate the integrity of the cartilage. We cing hyaline-like cartilage regeneration at 12 weeks post- also found that cartilage-associated genes (collagen type II, surgery in a sheep model [16]. Biomechanical testing is also AggC, Sox 9) showed high gene expression after 8 weeks. appropriate as the next step for tantalum-chondrocyte-fibrin This supports our hypothesis and suggested that the cartil- composite. age composite had produced a good matrix for the chon- In the present study, the tissue-engineered cartilage drocytes to differentiate. Similar findings were reported by showed morphological features of polygonal that became previous studies of human articular chondrocytes [15,23]. Figure 9 Immunohistochemical staining of in vivo construct shows strong reaction towards collagen type II (A) but weak reaction with collagen type I with brown discoloration extracellularly (B). Jamil et al. Journal of Orthopaedic Surgery and Research (2015) 10:27 Page 8 of 9 Figure 11 Comparison between in vivo constructs (4 and 8 weeks) to in vitro for the quantitative RT-PCR analysis (p<0.05) of gene expression of collagen type I (A) and collagen type X (B). Native isolated RNA from normal native cartilage. of a single surgery without the use of a periosteal patch which can be unstable. Figure 10 Comparison between in vivo constructs (4 and 8 weeks) to Conclusion in vitro for the quantitative RT-PCR analysis (p<0.05). It determined We showed that tantalum scaffold with fibrin as cell carrier the gene expression of collagen type II (A), aggrecan (B), and Sox 9 (C). promotes cellular proliferation and cartilaginous tissue Native isolated RNA from normal native cartilage. ormation of rabbit articular chondrocytes. Engineered car- tilage resulted from in vivo-implanted construct demon- Fibrocartilage and hypertrophic markers even though strated high-quality hyaline-like tissue by histological and present showed much lower expression level. Sasano et al. biochemical assessment, GAG quantification, immunohis- also discovered that chondrocytes synthesize collagen type tochemistry, and real-time PCR. This early results highlight I and accumulate the protein in the matrix during his the potential of tantalum-chondrocyte-fibrin composite in study of rat tibial articular cartilage [15,24]. treatment of cartilage defect. These findings are in accordance with previous studies Competing interests in proving that autologous chondrocyte and fibrin compos- The authors declare that they have no competing interests. ite produced good-quality cartilage-like tissue [21,25-32]. In Authors’ contributions addition, we overcome the concern of fibrin glue detach- KJ carried out the animal studies, participated in the laboratory assessments, ment and degradation by providing a good scaffold with and drafted the manuscript. SLN carried out the histological and biochemical tantalum. Tantalum usage has been well established in assessments, immunohistochemistry, PCR analysis, and statistical analysis. SJ participated in the cell preparation and harvest of composite. NHY conceived of clinical settings due to its biocompatibility and highly in- the study and participated in the design of the study. KHC conceived of the terconnected porosity which permits physiologic bone study, participated in its design and coordination, and helped to draft the ingrowth and healing [17,33-41]. By 8 weeks, this tantalum- manuscript. All authors read and approved the final manuscript. chondrocyte-fibrin composite show good promise in pro- Acknowledgements viding solution for cartilage defect. Clinically, this cartilage The source of funding for the study was from the Research Fund of construct may be implanted arthroscopically and remain in Universiti Kebangsaan Malaysia. We would like to thank Professor Srijit Das situ with a stable scaffold. This advantage offers the option for his help and contribution in completing the manuscript. Jamil et al. Journal of Orthopaedic Surgery and Research (2015) 10:27 Page 9 of 9 Author details 22. Ishak MF, Chua KH, Asma A, Saim L, Aminuddin BS, Ruszymah BH, et al. Department of Orthopaedic and Traumatology, Faculty of Medicine, Stem cell genes are poorly expressed in chondrocytes from microtic Universiti Kebangsaan Malaysia, Jalan Yaacob Latiff, Bandar Tun Razak, cartilage. Int J Pediatr Otorhinolaryngol. 2011;75:835–40. 56000 Cheras, Kuala Lumpur, Malaysia. Department of Physiology, Faculty of 23. Munirah S, Kim SH, Ruszymah BH, Khang G. The use of fibrin and poly Medicine, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, (lactic-co-glycolic acid) hybrid scaffold for articular cartilage tissue 50300 Kuala Lumpur, Malaysia. engineering: an in vivo analysis. Eur Cell Mater. 2008;15:41–52. 24. Sasano Y, Furusawa M, Ohtani H, Mizoguchi I, Takahashi I, Kagayama M. Received: 11 September 2014 Accepted: 14 January 2015 Chondrocytes synthesize type I collagen and accumulate the protein in the matrix during development of rat tibial articular cartilage. 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Journal of Orthopaedic Surgery and Research – Springer Journals

Published: Feb 7, 2015

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