The American Journal of Sports Medicine http://ajs.sagepub.com/
One-Step Repair for Cartilage Defects in a Rabbit Model: A Technique Combining the Perforated Decalcified Cortical-Cancellous Bone Matrix Scaffold With Microfracture Linghui Dai, Zhenming He, Xin Zhang, Xiaoqing Hu, Lan Yuan, Ming Qiang, Jingxian Zhu, Zhenxing Shao, Chunyan Zhou and Yingfang Ao Am J Sports Med 2014 42: 583 originally published online February 4, 2014 DOI: 10.1177/0363546513518415 The online version of this article can be found at: http://ajs.sagepub.com/content/42/3/583
Published by: http://www.sagepublications.com
On behalf of: American Orthopaedic Society for Sports Medicine
Additional services and information for The American Journal of Sports Medicine can be found at: Email Alerts: http://ajs.sagepub.com/cgi/alerts Subscriptions: http://ajs.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav
>> Version of Record - Feb 28, 2014 OnlineFirst Version of Record - Feb 4, 2014 What is This?
Downloaded from ajs.sagepub.com at DALHOUSIE UNIV on December 26, 2014
One-Step Repair for Cartilage Defects in a Rabbit Model A Technique Combining the Perforated Decalcified Cortical-Cancellous Bone Matrix Scaffold With Microfracture Linghui Dai,*y PhD, Zhenming He,* MD, Xin Zhang,* PhD, Xiaoqing Hu,* PhD, Lan Yuan,z PhD, Ming Qiang,§ BE, Jingxian Zhu,* PhD, Zhenxing Shao,* PhD, Chunyan Zhou,yk PhD, and Yingfang Ao,*k MD Investigation performed at Institute of Sports Medicine, Peking University Third Hospital, Beijing, PR China Background: Cartilage repair still presents a challenge to clinicians and researchers alike. A more effective, simpler procedure that can produce hyaline-like cartilage is needed for articular cartilage repair. Hypothesis: A technique combining microfracture with a biomaterial scaffold of perforated decalcified cortical-cancellous bone matrix (DCCBM; composed of cortical and cancellous parts) would create a 1-step procedure for hyaline-like cartilage repair. Study Design: Controlled laboratory study. Methods: For the in vitro portion of this study, mesenchymal stem cells (MSCs) were isolated from bone marrow aspirates of New Zealand White rabbits. Scanning electron microscopy (SEM), confocal microscopy, and 1,9-dimethylmethylene blue assay were used to assess the attachment, proliferation, and cartilage matrix production of MSCs grown on a DCCBM scaffold. For the in vivo experiment, full-thickness defects were produced in the articular cartilage of the trochlear groove of 45 New Zealand White rabbits, and the rabbits were then assigned to 1 of 3 treatment groups: perforated DCCBM combined with microfracture (DCCBM1M group), perforated DCCBM alone (DCCBM group), and microfracture alone (M group). Five rabbits in each group were sacrificed at 6, 12, or 24 weeks after the operation, and the repair tissues were analyzed by histological examination, assessment of matrix staining, SEM, and nanoindentation of biomechanical properties. Results: The DCCBM1M group showed hyaline-like articular cartilage repair, and the repair tissues appeared to have better matrix staining and revealed biomechanical properties close to those of the normal cartilage. Compared with the DCCBM+M group, there was unsatisfactory repair tissues with less matrix staining in the DCCBM group and no matrix staining in the M group, as well as poor integration with normal cartilage and poor biomechanical properties. Conclusion: The DCCBM scaffold is suitable for MSC growth and hyaline-like cartilage repair induction when combined with microfracture. Clinical Relevance: Microfracture combined with a DCCBM scaffold is a promising method that can be performed and adopted into clinical treatment for articular cartilage injuries. Keywords: articular cartilage; cartilage repair; microfracture; decalcified cortical-cancellous bone matrix (DCCBM); medical applications
Because of its limited capacity for self-repair, articular cartilage injury is a refractory disorder of joints. Many methods have been designed to promote the repair of cartilage, among which abrasion arthroplasty and osteochondral drilling is the most simple, widely used technique to reduce pain and improve joint function. However, its clinical effects last only a short time, and it achieves partial repair that leads to fibrocartilage formation.10,19,25 Autologous chondrocyte implantation has
Articular cartilage injury can affect people of all ages, resulting in severe pain, joint swelling, substantial reduction in mobility, and severe restrictions to one’s activities.
The American Journal of Sports Medicine, Vol. 42, No. 3 DOI: 10.1177/0363546513518415 Ó 2014 The Author(s)
583 Downloaded from ajs.sagepub.com at DALHOUSIE UNIV on December 26, 2014
584
Dai et al
The American Journal of Sports Medicine
been reported to successfully generate hyaline-like cartilage,7,28,32 but there are still many problems, such as insufficient cell supply, new damage to the donor site, and chondrocyte dedifferentiation during its expansion in vitro. Moreover, the patient always needs to undergo 2 operations to complete treatment. These problems also exist in other transplantation techniques, such as periosteal transplantation, osteochondral transplantation,18 and matrixassisted chondrocyte implantation.4,5 Clinically, the microfracture technique has become the first-line treatment for articular cartilage injury because it is relatively easy to perform, minimally invasive, and well tolerated by the patient.6,34 It allows marrow stromal cells containing mesenchymal stem cells (MSCs) to fill in the defect and participate in cartilage repair. The symptoms of patients can be relieved after microfracture. However, it also has some drawbacks: for instance, it can only produce fibrocartilage, and there is poor tissue filling of the defect, particularly around the margins, and poor integration with the native articular cartilage, especially in large cartilage defects.37 Therefore, a more effective, simpler procedure that can produce hyaline-like cartilage is needed for articular cartilage repair. Previously, Zhang et al39 demonstrated that the microfracture technique combined with perforated decalcified cortical bone matrix (DCBM) and adenovirus–bone morphogenetic protein 4 (Ad-BMP4) transfection successfully induced hyaline-like articular cartilage repair. The DCBM is a natural, 3-dimensional scaffold with good biocompatibility and with mechanical properties suitable for cartilage repair. However, having studied DCBM, we found that decalcification takes too much time, especially in larger animals such as pigs and goats. This is a potential setback to further research on larger animals and humans and clinical applications of the scaffold. As an improvement, in this study, we used a perforated decalcified cortical-cancellous bone matrix (DCCBM) instead; this graft is a compound of cortical bone and cancellous bone matrix. Compared with DCBM, it has a cancellous component, and the cortical component is thinner, thus increasing the porosity as well as the cell adhesion because the cancellous component is more porous than the cortical bone. In this study, we first evaluated the attachment of the MSCs on the scaffold in vitro. Then, we used the microfracture technique to recruit MSCs and combined the DCCBM scaffold to perform a hyaline-like cartilage in vivo. We established a novel strategy that simplifies the technical steps without adding any other exogenous growth factors and that can lead to cartilage repair in a single step.
MATERIALS AND METHODS We used New Zealand White rabbits for this study. All animals were purchased from Peking University Animal Administration Center. All animal experimental protocols were approved by the Animal Care and Use Committee of Peking University and were in compliance with the Guide for the Care and Use of Laboratory Animals (National Academies Press, National Institutes of Health Publication No. 85-23, revised 1996).
In Vitro Isolation and Evaluation of Rabbit Marrow MSCs For the in vitro experiment, MSCs were derived from bone marrow aspirates of the distal femur of 4-week-old rabbits using the reported method.39 Briefly, each aspirate was combined with 25 mL of MSCs in a growth medium consisting of 89% Dulbecco’s modified Eagle’s medium (DMEM)– low glucose (Gibco BRL/Life Technologies, Gaithersburg, Maryland, USA), 10% fetal bovine serum (HyClone Laboratories, Logan, Utah, USA), and 1% penicillin and 1% streptomycin (Gibco). The cells were incubated at 37°C with 5% humidified CO2. Nonadherent cells were removed by changing the culture medium after 3 days of incubation. After being cultured for 4 to 5 days, the cells reached confluence and were defined as passage 0. The cells used in subsequent experiments were passage 3. Scaffold Preparation. The DCCBM scaffolds were obtained from the iliac bone of allogeneic rabbits. The scaffold was demineralized by soaking in 0.5 M ethylenediaminetetraacetic acid (EDTA) at 4°C, pH 8.3, with fresh solution every day. The replaced EDTA solution was analyzed by atomic absorption spectrophotometry to track the demineralization process. The scaffolds were well dimineralized after about 7 days (see Appendix Figure A1 online at http://ajsm.sagepub.com/supplemental). The decalcified scaffolds were stored at –80°C. Cell Seeding on Scaffolds. For the cell seeding, the scaffolds were first cut into small pieces (5 3 5 3 5 mm) and then sterilized with cobalt-60 for 24 hours, soaked in 75% alcohol for 2 hours, washed in sterile phosphate-buffered saline (PBS) 3 times each for 10 minutes, and conditioned with DMEM overnight. To seed the scaffolds, a 20-mL cell suspension containing 1 3 105 rabbit MSCs was loaded onto the scaffold. After 1 hour for cell attachment, the seeded scaffolds were cultured in 1 mL DMEM containing 0.1 mM dexamethasone, 50 mg/mL ascorbate 2-phosphate, 40 mg/mL L-proline, 100 mg/mL sodium pyruvate, 1 ITS
k Address correspondence to Yingfang Ao, Institute of Sports Medicine, Peking University Third Hospital, 49 North Garden Rd, Haidian District, Beijing 100191, PR China (e-mail: [email protected], [email protected]); and Chunyan Zhou, Department of Biochemistry and Molecular Biology, Peking University School of Basic Medical Sciences, 38 Xueyuan Rd, Haidian District, Beijing 100191, PR China (e-mail: [email protected]). *Institute of Sports Medicine, Peking University Third Hospital, Haidian District, Beijing, PR China. y Department of Biochemistry and Molecular Biology, Peking University School of Basic Medical Sciences, Beijing, PR China. z Department of Medical and Health Analysis Center, Peking University Medical and Health Analysis Center, Beijing, PR China. § Department of Mechanics and Aerospace Engineering, College of Engineering Peking University, Beijing, PR China. Linghui Dai and Zhenming He contributed equally to this study. One or more of the authors has declared the following potential conflict of interest or source of funding: This work was supported by National Natural Science Foundation of China (NFSC; grants 81101390 and 81071474), Specialized Research Fund for the Doctoral Program of Higher Education (20110001130001 and 20090001120117), Leading Academic Discipline Project of Beijing Education Bureau, and the 111 Project of China (B07001).
Downloaded from ajs.sagepub.com at DALHOUSIE UNIV on December 26, 2014
Vol. 42, No. 3, 2014
One-Step Repair Technique for Cartilage Defects
supplement (6.25 mg/mL insulin, 6.25 mg/mL transferrin, and 6.25 ng/mL selenous acid), 1.25 mg/mL bovine serum albumin, and 5.35 mg/mL linoleic acid with 10 ng/mL transforming growth factor (TGF)–b3 at 37°C with 5% humidified CO2 for 1, 4, and 7 days. Culture medium was changed every 3 days. Sulfated GAG and DNA Quantification. Scaffold samples (with or without cells) were digested for 16 hours with papain cocktail (125 mg/mL papain, 5 mM L-cysteine, 100 mM Na2HPO4, 5 mM EDTA, pH 6.2) at 60°C for DNA and glycosaminoglycan (GAG) estimation. The DNA content was measured using the PicoGreen DNA assay kit (Invitrogen, Carlsbad, California, USA) as per the manufacturer’s protocol. In brief, 20 mL of the sample was mixed with 200 mL of Quant-iT PicoGreen reagent (1:200 dilution). The excitation and emission wavelengths of 480 and 528 nm, respectively, were measured using a fluorimeter. Readings were compared with standard curves made from calf thymus DNA (Sigma, St Louis, Missouri, USA).2 The DNA content was normalized to scaffold wet weight. Total sulfated GAG (sGAG) was estimated using the 1,9-dimethylmethylene blue (DMMB) assay.1 Briefly, 20 mL of sample was mixed with 200 mL of DMMB reagent, and absorbance was read on a plate reader (MultiSkan Spectrum; Thermo, Waltham, Massachusetts, USA) at 525 nm. A standard curve was established from chondroitin-6-sulfate from shark (Sigma) to compare absorbance for the samples.3 Total sGAG was normalized to scaffold wet weight and total DNA content to avoid variation from scaffold sizes and cell numbers. Confocal Microscopy. The scaffold samples (with cells) were washed 3 times with PBS (pH 7.4) and incubated with 0.1% (w/v) acridine orange for 5 minutes. Images from stained scaffolds were obtained using a confocal laser scanning microscope (Leica SP2 inverted microscope; Leica, Mannheim, Germany) equipped with 488-nm lasers.
In Vivo Animal Experiments The in vivo experiments were carried out based on previously reported methods.39 Forty-five New Zealand White rabbits (total 90 knees) weighing 2.5 to 3.0 kg (age, 4-6 months) were used for this part of the investigation. Rabbits were divided into 3 treatment groups (the 2 knees of each rabbit were used in the same group): microfracture alone (M group), DCCBM implantation alone (DCCBM group), and microfracture combined with DCCBM implantation (DCCBM1M group). The rabbits were anesthetized by intravenous injection of 10 mL ethylcarbamate (0.2 g/mL). After shaving, disinfection, and draping, the knee was opened by an anteromedial parapatellar incision and the patella was everted. Full-thickness articular defects, 4 mm in diameter, were created by corneal trephine in the trochlear groove of the distal femur. One of the following procedures was then performed: (1) DCCBM scaffold preparation: to allow bone marrow to penetrate DCCBM, holes were drilled through the DCCBM with a drill bit 0.5 mm in diameter. The drilled DCCBM was made into a cylinder 4 mm in diameter by a corneal trephine. (2) Microfracture: a microfracture
585
was made into the medullary cavity. (3) The scaffold was implanted into the defect by placing the cortical part of the scaffold in contact with the subchondral bone. All implants were placed at the same level with the surface of the adjacent cartilage. Wound closure was performed in layers. All rabbits were kept in cages and had free access to food pellets and water. At 6, 12, or 24 weeks after surgery, the rabbits were sacrificed with an intravenous injection of 5 mL pentobarbital (n = 5 rabbits [10 knees] in each group at each time point). Histological Assessment and Staining of Repair Tissue. After sacrifice, the distal portions of the femurs (6 samples in 3 rabbits in each group) were cut off and then fixed in 4% paraformaldehyde (pH 7.4) for 48 hours at 4°C. The femurs were decalcified in 20% EDTA (pH 7.2) in PBS with 5% paraformaldehyde at 4°C. The decalcified specimens were trimmed, dehydrated in a graded ethanol series, and embedded in paraffin. Serial sections (8 mm thick) were cut sagittally through the center of the operative site and stained with hematoxylin and eosin and toluidine blue. Immunohistochemistry was performed with type II collagen antibody (Abcam, Cambridge, UK). Histological Scoring System. A modified method36 was used to evaluate the histological repair of articular cartilage defects. The parameters include 7 categories: cell structure (hyaline cartilage), metachromatic staining of the cartilage matrix, structural integrity of the regenerated cartilage, surface regularity of the tissue, thickness of the cartilage layer, regeneration of the subchondral bone, and integration of the tissue with adjacent cartilage. The scores ranged from 0 to 18 points (see the online Appendix). Higher scores indicate better repairs. Three blinded reviewers were used to evaluate the scores. Scanning Electron Microscopy. The repair tissues were harvested 24 weeks from the surface of the implant in the trochlear groove of the right distal femur. The samples were fixed immediately in a mixture of 4 mL 25% glutaraldehyde and 96 mL 10 mmol/L Tris-HCl (pH 7.4), 4°C for 1 day, then dehydrated with a graded series of 70%, 80%, 90%, and 100% ethanol. Critical point drying was performed in liquid CO2 at 37°C. The specimens were vacuum-coated with a 5-nm layer of gold in a high-vacuum gold spatter coater and then viewed with a scanning electron microscope (S-2500; Hitachi High-Technologies Co, Hitachi-Naka City, Japan). Nanoindentation Assessment of Repair Tissue. The biomechanical analysis of the repair tissues was performed using the nanoindentation based on the reported methods.15,16,23 The samples (n = 9 each group) were isolated from the central part of the repair tissues. The normal hyaline cartilage samples (n = 9) were isolated from the nonoperated trochlear parts of the knee. The circumfluent PBS is to maintain hydration of the samples at room temperature during testing. All indentations were performed using the TriboIndenter (Hysitron Inc, Minneapolis, Minnesota, USA) with a 100-mm radius of a curvature conospherical diamond probe tip. For each indentation, the maximum indentation depth was set to 1000 nm, and the sample was loaded for the first 10 seconds once the tips contacted the
Downloaded from ajs.sagepub.com at DALHOUSIE UNIV on December 26, 2014
586
Dai et al
The American Journal of Sports Medicine
Figure 1. The proliferation and the matrix production of mesenchymal stem cells (MSCs) seeded on the decalcified cortical-cancellous bone matrix (DCCBM) scaffolds. (A) The DCCBM scaffold. (B) The proliferation of MSCs was quantitatively analyzed based on the ratio of total DNA/wet weight of the scaffold. (C and D) The matrix production of MSCs seeded on DCCBM is quantified by the ratio of sulfated glycosaminoglycan (sGAG)/wet weight of scaffold (C) and the ratio of sGAG/DNA content (D) at 1, 4, or 7 days. The MSCs continued to proliferate and produce sGAG with time. Data represent mean 6 standard deviation (n = 6; *P \ .05 vs 1 day). surface of the samples, held at the maximum depth for 2 seconds, and unloaded in the last 10 seconds. The data analysis of nanoindentation was performed based on the reported method.29,30,33 Details are in the online Appendix.
Statistical Analysis For statistical analysis, the statistical significance of the differences between groups was calculated using the KruskalWallis test of analysis of variance (ANOVA). The results from the same group were evaluated using a Student t test. P \ .05 was considered to indicate statistical significance. All results are expressed as mean 6 standard deviation.
RESULTS In Vitro Isolation and Assessment of MSCs Proliferation and Cartilage Matrix Production of MSCs Grown on the DCCBM Scaffold. The DCCBM scaffold used is shown in Figure 1A. We found that both DNA and sGAG content were increased with time (day 7 vs day 1: P = .0001 for DNA, P = .003 for sGAG) (Figure 1, B and C), indicating the MSCs were proliferated from day 1 to day 7 and continued to produce sGAG (cartilaginous matrix) during the
Figure 2. The attachment of mesenchymal stem cells (MSCs) on different parts of the decalcified corticalcancellous bone matrix (DCCBM) scaffold. Scanning electron microscopy shows the growth of MSCs on the surface of cortical parts (A-D) and cancellous parts (E-H) of the DCCBM at different magnifications (original magnification: A and E: 3200, B and F: 3500, C and G: 31000, D and H: 32000). Arrows indicate the typical cells grown on different parts of the scaffold. Confocal laser microscopic images show the growth of MSCs on the cortical parts of the DCCBM (I-L) and the cancellous parts (M-P). The MSCs grown on both parts of the scaffold were stained with green and red fluorescence uniformly (no apoptotic or necrotic cells). Green: the intercalated DNA by acridine orange with fluorescence at 515 to 545 nm. Red: the electrostatic RNA by acridine orange with fluorescence at 590 to 620 nm. Yellow: merged from I and J or M and N. Magnification: 360.
7 days of culture. Also, sGAG/DNA was significantly increased on day 7 compared with day 1 (P = .003) (Figure 1D), indicating that the ability of MSCs to produce sGAG, helpful for cartilage repair, increased after 7 days of culture. Attachment of MSCs Grown on the DCCBM Scaffold. The MSCs were well grown on both parts of the DCCBM scaffold according to images from the scanning electron microscope (SEM; JSM-5600LV; JEOL, Tokyo, Japan). The MSCs spread well (Figure 2, D and H) and adhered tightly to the cortical parts (Figure 2, A-D) and cancellous parts (Figure 2, E-H) of the scaffold, especially in the concave area of the DCCBM scaffold (Figure 2, B and F). Viability of MSCs Grown on the DCCBM Scaffold. A vital dye, acridine orange, was used to assess the viability of MSCs grown on the different parts of the DCCBM. Acridine orange interacts with DNA (515-545 nm) and RNA (590-620 nm). It can distinguish normal, apoptotic, and
Downloaded from ajs.sagepub.com at DALHOUSIE UNIV on December 26, 2014
Vol. 42, No. 3, 2014
One-Step Repair Technique for Cartilage Defects
587
Figure 3. Macroscopic appearance of the cartilage defect healing in 3 groups at 6 (A-C), 12 (D-F), and 24 (G-I) weeks after the operation. The 3 groups are as follows: M group, cartilage defects were treated with the microfracture technique alone; DCCBM group, cartilage defects were implanted with decalcified cortical-cancellous bone matrix (DCCBM) scaffold alone; and DCCBM1M group, cartilage defects were treated with microfracture and implanted with the DCCBM scaffold. necrotic cells. In normal cells, the nucleus was stained green or yellow-green; in apoptotic cells, the nucleus was densely stained with yellow-green fluorescence or yellow-green debris particles; in necrotic cells, the yellow fluorescence weakened or even disappeared. In the current study, the nuclei of MSCs grown on both cortical and cancellous parts were stained with green fluorescence uniformly (Figure 2, I and M). The MSCs were stained with red fluorescence uniformly, indicating that the cytoplasm of the MSCs was uniform, with no rupture and thus no apoptotic cells (Figure 2, J and N). The orientation and the shape of the cells were different. The MSCs in the cortical part appeared as fibroblastic shapes; however, in the cancellous part, they emerged as polygonal shapes, which may have resulted because the cell growth followed the texture of the scaffold. No apoptotic or necrotic cells were seen on these images (Figure 2, I-P).
In Vivo Treatment Groups Macroscopic Findings of the Repaired Cartilage Defects
At 6 Weeks. Repair tissues in the DCCBM1M group had well-filled cartilage defects, many more cells than normal cartilage, better integration, and an irregular surface (Figures 3C and 4C). Small parts (about 2 mm, 50%-60% depth of the defects region) of the cartilage defects were filled
Figure 4. Hematoxylin and eosin staining in 3 groups at 6 (A-C), 12 (D-F), and 24 (G-I) weeks after the operation. (J, K, L) Images of the repair tissues at 24 weeks in each group. (M) Image of the unrepaired cartilage defect with no treatment at 24 weeks. The repair tissues in the DCCBM1M group had a better performance than the other groups. DCCBM, decalcified cortical-cancellous bone matrix; DCCBM1M, perforated DCCBM combined with microfracture; N, normal cartilage; R, repair tissues. The arrows indicate the margins of the normal tissue and the repaired tissue. Black scale bars = 500 mm; white scale bars = 100 mm.
with the repair tissues, with no integration, and clear edges could be seen in the M group and DCCBM group (Figure 3, A and B). In the M group, the repair tissues were thinner and less complete than in the other 2 groups, and the subchondral bone in the defects area was discontinuous (Figure 4A). Occasional cell mass growth could be seen on the scaffold and the edge of normal cartilage in the DCCBM group (Figure 4B). At 12 Weeks. Cartilage defects were well filled with the same repair tissues as that at 6 weeks in the DCCBM1M group. Repair tissues were at the same height of the normal cartilage, and the surface was smoother than at 6 weeks (Figures 3F and 4F). The other 2 groups had improved repair tissues compared with that at 6 weeks, but these tissues were thinner, with no integration
Downloaded from ajs.sagepub.com at DALHOUSIE UNIV on December 26, 2014
588
Dai et al
The American Journal of Sports Medicine
Figure 5. Toluidine blue staining of repaired tissue and normal cartilage in 3 groups at 6 (A-C), 12 (D-F), and 24 (G-I) weeks after the operation. DCCBM, decalcified corticalcancellous bone matrix; DCCBM1M, perforated DCCBM combined with microfracture; N, normal cartilage; R, repair tissues. The arrows indicate the margins of the normal tissue and the repaired tissue. Scale bars = 500 mm. between the regenerated and the normal tissues (Figure 3, D and E). Cracks could be seen between the repaired and the normal tissues in the DCCBM group (Figure 4E). At 24 Weeks. In the DCCBM1M group, repair tissues fully filled in the cartilage defects and had a smooth surface; they were the same as the normal tissue in height, number, and arrangement of the cells (Figures 3I and 4I); moreover, the cell types were hyaline-like chondrocytes (Figure 4L). There was no edge on gross observation (Figure 3I), but little cracks in the hematoxylin and eosin staining (Figure 4I) could be seen between the repair tissues and the normal tissues. The tidemark was clear. However, cartilage defects were smaller than those at 6 and 12 weeks but not fully filled with repair tissues; incomplete integration, an uneven surface, and thinner repair tissues could also be seen in the M group and DCCBM group (Figures 3G, 3H, 4G and 4H); cells were arranged in a haphazard manner (Figure 4, J and K).
Assessment of Cartilage Matrix Production of the Repair Tissues The repair tissues of the M group had no staining with toluidine blue at 6 weeks; the other 2 groups received lighter staining with toluidine blue compared with normal cartilage (Figure 5, A-C). However, lighter staining with toluidine blue and no differences among the 3 groups could be seen at 12 weeks in the repair tissues (Figure 5, D-F). The repair tissues in the DCCBM1M group, which had uniform toluidine blue staining, were much closer to the normal cartilage at 24 weeks (Figure 5I). The other 2 groups had lighter staining compared with normal cartilage at 24 weeks (Figure 5, G and H).
Figure 6. The type II collagen immunohistological staining of repaired tissue and normal cartilage in 3 groups at 6 (A-C), 12 (D-F), and 24 (G-I) weeks after the operation. DCCBM, decalcified cortical-cancellous bone matrix; DCCBM1M, perforated DCCBM combined with microfracture; N, normal cartilage; R, repair tissues. The arrows indicate the margins of the normal tissue and the repaired tissue. Scale bars = 500 mm. Similar to the staining with toluidine blue, staining with collagen type II was especially stronger in the DCCBM1M group compared with the other 2 groups at 6, 12, and 24 weeks after operation and implantation (Figure 6).
Histological Scores The histological scores of the 3 groups are consistent with the results shown above. The score in the DCCBM1M group increased with time (from 10.80 6 2.22 to 12.40 6 2.37, finally increasing to 14.90 6 1.35 at 6, 12, and 24 weeks, respectively). However, little change was seen in the other 2 groups (DCCBM group: from 7.7 6 2.60 to 9.10 6 2.50, finally decreasing to 8.30 6 2.60; M group: from 6.60 6 1.80 to 6.90 6 2.34, finally increasing to 7.30 6 2.19). There were higher scores for repair tissues in the DCCBM1M group compared with the M group (P = .0001) at 12 weeks and compared with both the M group (P = .0001) and DCCBM group (P = .02) at 24 weeks. In addition, the repair tissues in the DCCBM1M group at 24 weeks received higher scores than at 6 weeks (P = .0001) (Figure 7). No such trend was seen in the other 2 groups, indicating that the repaired effects improved with longer duration of implantation.
Assessment of the Integration Between the Normal and Repair Tissues There were cracks between the repair tissues and the normal tissues in the M group and the DCCBM group, although the repair tissues in the DCCBM group were smoother than those in the M group (Figure 8, A and B). However, the edges between the repair tissues and normal
Downloaded from ajs.sagepub.com at DALHOUSIE UNIV on December 26, 2014
Vol. 42, No. 3, 2014
One-Step Repair Technique for Cartilage Defects
589
Figure 7. Histological assessment according to the method described by Wakitani et al36 for the repair tissues in the 3 groups. Higher scores can be seen in DCCBM1M group. DCCBM, decalcified cortical-cancellous bone matrix; DCCBM1M, perforated DCCBM combined with microfracture; M, microfracture alone. *P \ .05.
Figure 8. Scanning electron microscopy images from 3 groups at 24 weeks after the operation. DCCBM, decalcified cortical-cancellous bone matrix; DCCBM1M, perforated DCCBM combined with microfracture; M, microfracture alone; N, normal cartilage; R, repair tissues. A smooth surface can be seen in the repair tissues of the DCCBM1M group. The arrows indicate the margins of the normal and repaired tissue. Scale bars = 500 mm. tissues were not integrated in either the M or the DCCBM group. In the DCCBM1M group, the surface of the repair tissues was smooth, at the same height of the normal cartilage, and well integrated (Figure 8C).
Assessment of the Biomechanical Properties of the Repair Tissues The repair tissues were cut into small round pieces (diameter = 4 mm) for biomechanical analysis (Figure 9A). The typical load-displacement curves in the 3 groups were compared with those in the normal cartilage (Figure 9B). The contact stiffness is considered a suitable measurement for comparing the quality of different repair tissues. Reduced
Figure 9. Mechanical properties of the normal cartilage and the repaired tissue. (A) Samples from the central part of the repaired tissue 24 weeks after transplantation were evaluated by a nanoindenter. (B) Typical load-displacement curves in tissues from 3 groups were compared with the normal cartilage load-displacement curves, providing the tissue response to contact deformation. Contact stiffness (C), reduced modulus (D), and hardness (E) of the repair tissues were compared with the normal cartilage. Higher mechanical properties can be seen in the repair tissues in the DCCBM1M group. DCCBM, decalcified cortical-cancellous bone matrix; DCCBM1M, perforated DCCBM combined with microfracture; M, microfracture alone; N, normal cartilage. (n = 9, *P \ .05) modulus, which represents the elastic deformation that occurs in both sample and indenter tips, is more sensitive to subtle changes in the mechanical behavior. These parameters have been used as key properties in the assessment of cartilage mechanical properties by nanoindentation.15,16,23 In our study, normal cartilage had the highest contact stiffness, followed, in descending order of stiffness, by the samples from the DCCBM1M, DCCBM, and M groups (Figure 9C). Similar results can be seen in the assessment of reduced modulus and hardness (Figure 9, D and E). Normal cartilage displayed the highest contact stiffness, as well as reduced modulus and hardness, while the repair tissues in the M group had the lowest stiffness among the 3 groups (Figure 9, C-E). However, there were
Downloaded from ajs.sagepub.com at DALHOUSIE UNIV on December 26, 2014
590
Dai et al
The American Journal of Sports Medicine
no statistically significant differences between the DCCBM1M group and the normal cartilage in contact stiffness and reduced modulus (Figure 9, C and D); the other groups had significantly lower results compared with normal cartilage (contact stiffness: normal cartilage vs M group, P \ .001, and normal cartilage vs DCCBM group, P \ .001; reduced modulus: normal cartilage vs M group, P \ .001, and normal cartilage vs DCCBM group, P \ .001). These findings may indicate that the biomechanical properties of repaired tissue in the DCCBM1M group were close to those of normal cartilage.
DISCUSSION Recently, single-step repair methods for cartilage repair have been developed to avoid cell manipulation and multiple surgical procedures.11,17 To achieve 1-step cartilage repair, obtaining the cell source is the primary task that needs to be solved. The microfracture technique is a simple method to solve this problem. It can induce MSCs and the chondroprogenitors to migrate into the cartilage lesion and can enhance cytokine release that promotes cartilage repair.24 This method is commonly used in clinics for articular cartilage repairs.22 However, as already reported,26 the cartilage defects usually lead to a fibrocartilage repair. This is probably due to the limited number of cells from the bone marrow, thus making it difficult to induce hyaline cartilage formation.35 However, the surgical approach of creating a microfracture to induce endogenous stem cells seems more favorable than exogenous stem cells in the repair strategy. Also, microfracture can relieve pain, is easy to perform, and can save money for the patient. Therefore, many scaffolds were designed to create a suitable microenvironment to induce MSCs and progenitor cells to acquire chondrocyte-like properties. Previously, native hyaline-like articular cartilage repair was successfully induced by combining microfracture, DCBM, and Ad-BMP4.39 However, the process of the decalcification of DCBM takes too much time because the cortical bone matrix is not porous enough. The use of DCCBM successfully solved this problem. The DCCBM scaffold is more porous (see Appendix Figure A2) and has natural ingredients appropriate for cell growth and the induction of cartilage. The use of a viral vector is always a concern for clinical application.9,38 In the present study, we demonstrated that without the use of Ad-BMP4, the optimized scaffold DCCBM combined with microfracture could also induce a hyalinelike articular cartilage repair. This method is simpler and can avoid the shortcomings of using gene therapy. Moreover, we found that a hyaline-like articular cartilage repair could be observed only in the DCCBM1M group. In the DCCBM-alone group, although the DCCBM scaffold could fill the cartilage defects and the cells could grow on the scaffold, the repair tissues did not appear to have a smooth surface; the matrix staining and the biomechanical properties of repair tissues were weaker than the normal cartilage and the tissues in the DCCBM1M group. In the M group, similar to the other reported studies, the repair tissues were more like a fibrous scar rather than like cartilage.
There are studies that have focused on using MSCs and scaffolds for cartilage repair.8,27,31 Among them, autologous or allogeneic MSCs, growth factors (such as transforming growth factors and bone morphogenetic proteins), and scaffolds are the necessary components, and some require 2 operations to get a thorough regeneration of cartilage. However, our technique is a cell-free, 1-step procedure to get a better regeneration of cartilage. Several factors may contribute to the better repair results. First, the cells induced by the microfracture can migrate to the cartilage defect region. Second, the porous structure of the DCCBM scaffold is conducive to increased cell growth. This is also one of the reasons why the DCCBM scaffold is better than the DCBM scaffold. Third, the DCCBM scaffold itself contains many proteins that may be helpful for the proliferation and differentiation of cells. And fourth, the scaffold promoted cellular production of sGAG, one of the components of cartilage matrix in vitro. Recently, several studies have focused on the 1-step procedure for cartilage repair using biomaterials, such as poly(lactic-co-glycolic) acid (PLGA),12 nanomaterials,21 and hydrogel.20 These are all synthetic materials, and some have not been approved for clinical use. The DCCBM scaffold is made of decalcified bone matrix, a product of processed allograft bone. This is a natural material that has good biocompatibility with the human body and contains proteins that may be helpful for cells compared with other biomaterials. It is easier to get from tissue banks or donors, has been widely used worldwide to repair bone defects, and has been approved for clinical application by the United States Food and Drug Administration. The difference is that the scaffold used in repairing bone defects has not been drilled and may not include the cancellous part of the bone. Because of its ability to measure mechanical properties in situ without disrupting the complex microstructure,14 nanoindentation has been used to measure functional mechanical properties of repaired cartilage in a rabbit knee.13,16 In the present study, contact stiffness, reduced modulus, and hardness were used to assess the biomechanical properties of repair tissues via the nanoindentation technique. However, the indentation modulus is not a constant value in an inhomogeneous tissue such as articular cartilage and is highly dependent on the indentation depth and other factors, such as hydration.16 The modulus values in this study are relative values that aim to compare the different repair tissues under our identical testing conditions. These values may not be simply compared with the results of other reports, because both contact stiffness and reduced modulus are influenced by various methodological and environmental factors.
CONCLUSION The current study offers a promising way to improve 1-step cartilage repair by combining microfracture with the DCCBM scaffold. These findings demonstrate that this biotechnology is easy to perform and leads to hyaline-like cartilage repair in a rabbit model. Long-term studies in other animals as well as in patients are currently in progress.
Downloaded from ajs.sagepub.com at DALHOUSIE UNIV on December 26, 2014
Vol. 42, No. 3, 2014
One-Step Repair Technique for Cartilage Defects
REFERENCES 1. Abedi G, Sotoudeh A, Soleymani M, Shafiee A, Mortazavi P, Aflatoonian MR. A collagen-poly(vinyl alcohol) nanofiber scaffold for cartilage repair [published online December 1, 2010]. J Biomater Sci Polym Ed. 2. Adkisson HD 4th, Martin JA, Amendola RL, et al. The potential of human allogeneic juvenile chondrocytes for restoration of articular cartilage. Am J Sports Med. 2010;38(7):1324-1333. 3. Autologous chondrocytes. Autologous chondrocyte implantation: more data needed. Prescrire Int. 2011;20(116):122-124. 4. Bartlett W, Skinner JA, Gooding CR, et al. Autologous chondrocyte implantation versus matrix-induced autologous chondrocyte implantation for osteochondral defects of the knee: a prospective, randomised study. J Bone Joint Surg Br. 2005;87(5):640-645. 5. Behrens P, Bitter T, Kurz B, Russlies M. Matrix-associated autologous chondrocyte transplantation/implantation (MACT/MACI)—5year follow-up. Knee. 2006;13(3):194-202. 6. Bekkers JE, Inklaar M, Saris DB. Treatment selection in articular cartilage lesions of the knee: a systematic review. Am J Sports Med. 2009;37(suppl 1):148S-155S. 7. Beris AE, Lykissas MG, Kostas-Agnantis I, Manoudis GN. Treatment of full-thickness chondral defects of the knee with autologous chondrocyte implantation: a functional evaluation with long-term followup. Am J Sports Med. 2012;40:562-567. 8. Berninger MT, Wexel G, Rummeny EJ, et al. Treatment of osteochondral defects in the rabbit’s knee joint by implantation of allogeneic mesenchymal stem cells in fibrin clots. J Vis Exp. 2013;(75):e4423. 9. Bouard D, Alazard-Dany D, Cosset FL. Viral vectors: from virology to transgene expression. Br J Pharmacol. 2009;157(2):153-165. 10. Bouwmeester PS, Kuijer R, Homminga GN, Bulstra SK, Geesink RG. A retrospective analysis of two independent prospective cartilage repair studies: autogenous perichondrial grafting versus subchondral drilling 10 years post-surgery. J Orthop Res. 2002;20(2):267-273. 11. Buda R, Vannini F, Cavallo M, Grigolo B, Cenacchi A, Giannini S. Osteochondral lesions of the knee: a new one-step repair technique with bone-marrow-derived cells. J Bone Joint Surg Am. 2010;92(suppl 2):2-11. 12. Chang NJ, Lin CC, Li CF, Wang DA, Issariyaku N, Yeh ML. The combined effects of continuous passive motion treatment and acellular PLGA implants on osteochondral regeneration in the rabbit. Biomaterials. 2012;33(11):3153-3163. 13. Ebenstein DM, Kuo A, Rodrigo JJ, Reddi AH, Ries M, Pruitt L. A nanoindentation technique for functional evaluation of cartilage repair tissue. J Mater Res. 2004;19(1):273-281. 14. Ebenstein DM, Pruitt LA. Nanoindentation of biological materials. Nanotoday. 2006;1(3):26-33. 15. Franke O, Durst K, Maier V, et al. Mechanical properties of hyaline and repair cartilage studied by nanoindentation. Acta Biomater. 2007;3(6):873-881. 16. Gelse K, Muhle C, Franke O, et al. Cell-based resurfacing of large cartilage defects: long-term evaluation of grafts from autologous transgene-activated periosteal cells in a porcine model of osteoarthritis. Arthritis Rheum. 2008;58(2):475-488. 17. Giannini S, Buda R, Vannini F, Cavallo M, Grigolo B. One-step bone marrow–derived cell transplantation in talar osteochondral lesions. Clin Orthop Relat Res. 2009;467(12):3307-3320. 18. Hangody L, Dobos J, Balo E, Panics G, Hangody LR, Berkes I. Clinical experiences with autologous osteochondral mosaicplasty in an athletic population: a 17-year prospective multicenter study. Am J Sports Med. 2010;38(6):1125-1133. 19. Johnson LL. Arthroscopic abrasion arthroplasty: a review. Clin Orthop Relat Res. 2001;391(suppl):S306-S317.
591
20. Kitamura N, Yasuda K, Ogawa M, et al. Induction of spontaneous hyaline cartilage regeneration using a double-network gel: efficacy of a novel therapeutic strategy for an articular cartilage defect. Am J Sports Med. 2011;39(6):1160-1169. 21. Kon E, Delcogliano M, Filardo G, Busacca M, Di Martino A, Marcacci M. Novel nano-composite multilayered biomaterial for osteochondral regeneration: a pilot clinical trial. Am J Sports Med. 2011;39(6):1180-1190. 22. Kreuz PC, Steinwachs MR, Erggelet C, et al. Results after microfracture of full-thickness chondral defects in different compartments in the knee. Osteoarthritis Cartilage. 2006;14(11):1119-1125. 23. Li C, Pruitt LA, King KB. Nanoindentation differentiates tissue-scale functional properties of native articular cartilage. J Biomed Mater Res A. 2006;78(4):729-738. 24. Lotz M. Cytokines in cartilage injury and repair. Clin Orthop Relat Res. 2001;391(suppl):S108-S115. 25. Matsunaga D, Akizuki S, Takizawa T, Yamazaki I, Kuraishi J. Repair of articular cartilage and clinical outcome after osteotomy with microfracture or abrasion arthroplasty for medial gonarthrosis. Knee. 2007;14(6):465-471. 26. Miller BS, Steadman JR, Briggs KK, Rodrigo JJ, Rodkey WG. Patient satisfaction and outcome after microfracture of the degenerative knee. J Knee Surg. 2004;17(1):13-17. 27. Morille M, Van-Thanh T, Garric X, et al. New PLGA-P188-PLGA matrix enhances TGF-beta3 release from pharmacologically active microcarriers and promotes chondrogenesis of mesenchymal stem cells. J Control Release. 2013;170(1):99-110. 28. Moseley JB Jr, Anderson AF, Browne JE, et al. Long-term durability of autologous chondrocyte implantation: a multicenter, observational study in US patients. Am J Sports Med. 2010;38(2):238-246. 29. Oliver WC, Pharr GM. An improved technique for determining hardness and elastic-modulus using load and displacement sensing indentation experiments. J Mater Res. 1992;7(6):1564-1583. 30. Oliver WC, Pharr GM. Measurement of hardness and elastic modulus by instrumented indentation: advances in understanding and refinements to methodology. J Mater Res. 2004;19(1):3-20. 31. Park JS, Woo DG, Yang HN, Lim HJ, Chung HM, Park KH. Heparinbound transforming growth factor-beta3 enhances neocartilage formation by rabbit mesenchymal stem cells. Transplantation. 2008;85(4):589-596. 32. Peterson L, Vasiliadis HS, Brittberg M, Lindahl A. Autologous chondrocyte implantation: a long-term follow-up. Am J Sports Med. 2010;38(6):1117-1124. 33. Pharr GM, Oliver WC, Brotzen FR. On the generality of the relationship between contact stiffness, contact area, and elastic modulus during indentation. J Mater Res. 1992;7(3):613-617. 34. Schindler OS. Current concepts of articular cartilage repair. Acta Orthop Belg. 2011;77(6):709-726. 35. Steadman JR, Briggs KK, Rodrigo JJ, Kocher MS, Gill TJ, Rodkey WG. Outcomes of microfracture for traumatic chondral defects of the knee: average 11-year follow-up. Arthroscopy. 2003;19(5):477484. 36. Wakitani S, Goto T, Pineda SJ, et al. Mesenchymal cell-based repair of large, full-thickness defects of articular cartilage. J Bone Joint Surg Am. 1994;76(4):579-592. 37. Williams RJ 3rd, Harnly HW. Microfracture: indications, technique, and results. Instr Course Lect. 2007;56:419-428. 38. Young LS, Searle PF, Onion D, Mautner V. Viral gene therapy strategies: from basic science to clinical application. J Pathol. 2006;208(2):299-318. 39. Zhang X, Zheng Z, Liu P, et al. The synergistic effects of microfracture, perforated decalcified cortical bone matrix and adenovirusbone morphogenetic protein-4 in cartilage defect repair. Biomaterials. 2008;29(35):4616-4629.
For reprints and permission queries, please visit SAGE’s Web site at http://www.sagepub.com/journalsPermissions.nav
Downloaded from ajs.sagepub.com at DALHOUSIE UNIV on December 26, 2014
One-Step Repair for Cartilage Defects in a Rabbit Model: A Technique Combining the Perforated Decalcified Cortical-Cancellous Bone Matrix Scaffold With Microfracture Linghui Dai, Zhenming He, Xin Zhang, Xiaoqing Hu, Lan Yuan, Ming Qiang, Jingxian Zhu, Zhenxing Shao, Chunyan Zhou and Yingfang Ao Am J Sports Med 2014 42: 583 originally published online February 4, 2014 DOI: 10.1177/0363546513518415 The online version of this article can be found at: http://ajs.sagepub.com/content/42/3/583
Published by: http://www.sagepublications.com
On behalf of: American Orthopaedic Society for Sports Medicine
Additional services and information for The American Journal of Sports Medicine can be found at: Email Alerts: http://ajs.sagepub.com/cgi/alerts Subscriptions: http://ajs.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav
>> Version of Record - Feb 28, 2014 OnlineFirst Version of Record - Feb 4, 2014 What is This?
Downloaded from ajs.sagepub.com at DALHOUSIE UNIV on December 26, 2014
One-Step Repair for Cartilage Defects in a Rabbit Model A Technique Combining the Perforated Decalcified Cortical-Cancellous Bone Matrix Scaffold With Microfracture Linghui Dai,*y PhD, Zhenming He,* MD, Xin Zhang,* PhD, Xiaoqing Hu,* PhD, Lan Yuan,z PhD, Ming Qiang,§ BE, Jingxian Zhu,* PhD, Zhenxing Shao,* PhD, Chunyan Zhou,yk PhD, and Yingfang Ao,*k MD Investigation performed at Institute of Sports Medicine, Peking University Third Hospital, Beijing, PR China Background: Cartilage repair still presents a challenge to clinicians and researchers alike. A more effective, simpler procedure that can produce hyaline-like cartilage is needed for articular cartilage repair. Hypothesis: A technique combining microfracture with a biomaterial scaffold of perforated decalcified cortical-cancellous bone matrix (DCCBM; composed of cortical and cancellous parts) would create a 1-step procedure for hyaline-like cartilage repair. Study Design: Controlled laboratory study. Methods: For the in vitro portion of this study, mesenchymal stem cells (MSCs) were isolated from bone marrow aspirates of New Zealand White rabbits. Scanning electron microscopy (SEM), confocal microscopy, and 1,9-dimethylmethylene blue assay were used to assess the attachment, proliferation, and cartilage matrix production of MSCs grown on a DCCBM scaffold. For the in vivo experiment, full-thickness defects were produced in the articular cartilage of the trochlear groove of 45 New Zealand White rabbits, and the rabbits were then assigned to 1 of 3 treatment groups: perforated DCCBM combined with microfracture (DCCBM1M group), perforated DCCBM alone (DCCBM group), and microfracture alone (M group). Five rabbits in each group were sacrificed at 6, 12, or 24 weeks after the operation, and the repair tissues were analyzed by histological examination, assessment of matrix staining, SEM, and nanoindentation of biomechanical properties. Results: The DCCBM1M group showed hyaline-like articular cartilage repair, and the repair tissues appeared to have better matrix staining and revealed biomechanical properties close to those of the normal cartilage. Compared with the DCCBM+M group, there was unsatisfactory repair tissues with less matrix staining in the DCCBM group and no matrix staining in the M group, as well as poor integration with normal cartilage and poor biomechanical properties. Conclusion: The DCCBM scaffold is suitable for MSC growth and hyaline-like cartilage repair induction when combined with microfracture. Clinical Relevance: Microfracture combined with a DCCBM scaffold is a promising method that can be performed and adopted into clinical treatment for articular cartilage injuries. Keywords: articular cartilage; cartilage repair; microfracture; decalcified cortical-cancellous bone matrix (DCCBM); medical applications
Because of its limited capacity for self-repair, articular cartilage injury is a refractory disorder of joints. Many methods have been designed to promote the repair of cartilage, among which abrasion arthroplasty and osteochondral drilling is the most simple, widely used technique to reduce pain and improve joint function. However, its clinical effects last only a short time, and it achieves partial repair that leads to fibrocartilage formation.10,19,25 Autologous chondrocyte implantation has
Articular cartilage injury can affect people of all ages, resulting in severe pain, joint swelling, substantial reduction in mobility, and severe restrictions to one’s activities.
The American Journal of Sports Medicine, Vol. 42, No. 3 DOI: 10.1177/0363546513518415 Ó 2014 The Author(s)
583 Downloaded from ajs.sagepub.com at DALHOUSIE UNIV on December 26, 2014
584
Dai et al
The American Journal of Sports Medicine
been reported to successfully generate hyaline-like cartilage,7,28,32 but there are still many problems, such as insufficient cell supply, new damage to the donor site, and chondrocyte dedifferentiation during its expansion in vitro. Moreover, the patient always needs to undergo 2 operations to complete treatment. These problems also exist in other transplantation techniques, such as periosteal transplantation, osteochondral transplantation,18 and matrixassisted chondrocyte implantation.4,5 Clinically, the microfracture technique has become the first-line treatment for articular cartilage injury because it is relatively easy to perform, minimally invasive, and well tolerated by the patient.6,34 It allows marrow stromal cells containing mesenchymal stem cells (MSCs) to fill in the defect and participate in cartilage repair. The symptoms of patients can be relieved after microfracture. However, it also has some drawbacks: for instance, it can only produce fibrocartilage, and there is poor tissue filling of the defect, particularly around the margins, and poor integration with the native articular cartilage, especially in large cartilage defects.37 Therefore, a more effective, simpler procedure that can produce hyaline-like cartilage is needed for articular cartilage repair. Previously, Zhang et al39 demonstrated that the microfracture technique combined with perforated decalcified cortical bone matrix (DCBM) and adenovirus–bone morphogenetic protein 4 (Ad-BMP4) transfection successfully induced hyaline-like articular cartilage repair. The DCBM is a natural, 3-dimensional scaffold with good biocompatibility and with mechanical properties suitable for cartilage repair. However, having studied DCBM, we found that decalcification takes too much time, especially in larger animals such as pigs and goats. This is a potential setback to further research on larger animals and humans and clinical applications of the scaffold. As an improvement, in this study, we used a perforated decalcified cortical-cancellous bone matrix (DCCBM) instead; this graft is a compound of cortical bone and cancellous bone matrix. Compared with DCBM, it has a cancellous component, and the cortical component is thinner, thus increasing the porosity as well as the cell adhesion because the cancellous component is more porous than the cortical bone. In this study, we first evaluated the attachment of the MSCs on the scaffold in vitro. Then, we used the microfracture technique to recruit MSCs and combined the DCCBM scaffold to perform a hyaline-like cartilage in vivo. We established a novel strategy that simplifies the technical steps without adding any other exogenous growth factors and that can lead to cartilage repair in a single step.
MATERIALS AND METHODS We used New Zealand White rabbits for this study. All animals were purchased from Peking University Animal Administration Center. All animal experimental protocols were approved by the Animal Care and Use Committee of Peking University and were in compliance with the Guide for the Care and Use of Laboratory Animals (National Academies Press, National Institutes of Health Publication No. 85-23, revised 1996).
In Vitro Isolation and Evaluation of Rabbit Marrow MSCs For the in vitro experiment, MSCs were derived from bone marrow aspirates of the distal femur of 4-week-old rabbits using the reported method.39 Briefly, each aspirate was combined with 25 mL of MSCs in a growth medium consisting of 89% Dulbecco’s modified Eagle’s medium (DMEM)– low glucose (Gibco BRL/Life Technologies, Gaithersburg, Maryland, USA), 10% fetal bovine serum (HyClone Laboratories, Logan, Utah, USA), and 1% penicillin and 1% streptomycin (Gibco). The cells were incubated at 37°C with 5% humidified CO2. Nonadherent cells were removed by changing the culture medium after 3 days of incubation. After being cultured for 4 to 5 days, the cells reached confluence and were defined as passage 0. The cells used in subsequent experiments were passage 3. Scaffold Preparation. The DCCBM scaffolds were obtained from the iliac bone of allogeneic rabbits. The scaffold was demineralized by soaking in 0.5 M ethylenediaminetetraacetic acid (EDTA) at 4°C, pH 8.3, with fresh solution every day. The replaced EDTA solution was analyzed by atomic absorption spectrophotometry to track the demineralization process. The scaffolds were well dimineralized after about 7 days (see Appendix Figure A1 online at http://ajsm.sagepub.com/supplemental). The decalcified scaffolds were stored at –80°C. Cell Seeding on Scaffolds. For the cell seeding, the scaffolds were first cut into small pieces (5 3 5 3 5 mm) and then sterilized with cobalt-60 for 24 hours, soaked in 75% alcohol for 2 hours, washed in sterile phosphate-buffered saline (PBS) 3 times each for 10 minutes, and conditioned with DMEM overnight. To seed the scaffolds, a 20-mL cell suspension containing 1 3 105 rabbit MSCs was loaded onto the scaffold. After 1 hour for cell attachment, the seeded scaffolds were cultured in 1 mL DMEM containing 0.1 mM dexamethasone, 50 mg/mL ascorbate 2-phosphate, 40 mg/mL L-proline, 100 mg/mL sodium pyruvate, 1 ITS
k Address correspondence to Yingfang Ao, Institute of Sports Medicine, Peking University Third Hospital, 49 North Garden Rd, Haidian District, Beijing 100191, PR China (e-mail: [email protected], [email protected]); and Chunyan Zhou, Department of Biochemistry and Molecular Biology, Peking University School of Basic Medical Sciences, 38 Xueyuan Rd, Haidian District, Beijing 100191, PR China (e-mail: [email protected]). *Institute of Sports Medicine, Peking University Third Hospital, Haidian District, Beijing, PR China. y Department of Biochemistry and Molecular Biology, Peking University School of Basic Medical Sciences, Beijing, PR China. z Department of Medical and Health Analysis Center, Peking University Medical and Health Analysis Center, Beijing, PR China. § Department of Mechanics and Aerospace Engineering, College of Engineering Peking University, Beijing, PR China. Linghui Dai and Zhenming He contributed equally to this study. One or more of the authors has declared the following potential conflict of interest or source of funding: This work was supported by National Natural Science Foundation of China (NFSC; grants 81101390 and 81071474), Specialized Research Fund for the Doctoral Program of Higher Education (20110001130001 and 20090001120117), Leading Academic Discipline Project of Beijing Education Bureau, and the 111 Project of China (B07001).
Downloaded from ajs.sagepub.com at DALHOUSIE UNIV on December 26, 2014
Vol. 42, No. 3, 2014
One-Step Repair Technique for Cartilage Defects
supplement (6.25 mg/mL insulin, 6.25 mg/mL transferrin, and 6.25 ng/mL selenous acid), 1.25 mg/mL bovine serum albumin, and 5.35 mg/mL linoleic acid with 10 ng/mL transforming growth factor (TGF)–b3 at 37°C with 5% humidified CO2 for 1, 4, and 7 days. Culture medium was changed every 3 days. Sulfated GAG and DNA Quantification. Scaffold samples (with or without cells) were digested for 16 hours with papain cocktail (125 mg/mL papain, 5 mM L-cysteine, 100 mM Na2HPO4, 5 mM EDTA, pH 6.2) at 60°C for DNA and glycosaminoglycan (GAG) estimation. The DNA content was measured using the PicoGreen DNA assay kit (Invitrogen, Carlsbad, California, USA) as per the manufacturer’s protocol. In brief, 20 mL of the sample was mixed with 200 mL of Quant-iT PicoGreen reagent (1:200 dilution). The excitation and emission wavelengths of 480 and 528 nm, respectively, were measured using a fluorimeter. Readings were compared with standard curves made from calf thymus DNA (Sigma, St Louis, Missouri, USA).2 The DNA content was normalized to scaffold wet weight. Total sulfated GAG (sGAG) was estimated using the 1,9-dimethylmethylene blue (DMMB) assay.1 Briefly, 20 mL of sample was mixed with 200 mL of DMMB reagent, and absorbance was read on a plate reader (MultiSkan Spectrum; Thermo, Waltham, Massachusetts, USA) at 525 nm. A standard curve was established from chondroitin-6-sulfate from shark (Sigma) to compare absorbance for the samples.3 Total sGAG was normalized to scaffold wet weight and total DNA content to avoid variation from scaffold sizes and cell numbers. Confocal Microscopy. The scaffold samples (with cells) were washed 3 times with PBS (pH 7.4) and incubated with 0.1% (w/v) acridine orange for 5 minutes. Images from stained scaffolds were obtained using a confocal laser scanning microscope (Leica SP2 inverted microscope; Leica, Mannheim, Germany) equipped with 488-nm lasers.
In Vivo Animal Experiments The in vivo experiments were carried out based on previously reported methods.39 Forty-five New Zealand White rabbits (total 90 knees) weighing 2.5 to 3.0 kg (age, 4-6 months) were used for this part of the investigation. Rabbits were divided into 3 treatment groups (the 2 knees of each rabbit were used in the same group): microfracture alone (M group), DCCBM implantation alone (DCCBM group), and microfracture combined with DCCBM implantation (DCCBM1M group). The rabbits were anesthetized by intravenous injection of 10 mL ethylcarbamate (0.2 g/mL). After shaving, disinfection, and draping, the knee was opened by an anteromedial parapatellar incision and the patella was everted. Full-thickness articular defects, 4 mm in diameter, were created by corneal trephine in the trochlear groove of the distal femur. One of the following procedures was then performed: (1) DCCBM scaffold preparation: to allow bone marrow to penetrate DCCBM, holes were drilled through the DCCBM with a drill bit 0.5 mm in diameter. The drilled DCCBM was made into a cylinder 4 mm in diameter by a corneal trephine. (2) Microfracture: a microfracture
585
was made into the medullary cavity. (3) The scaffold was implanted into the defect by placing the cortical part of the scaffold in contact with the subchondral bone. All implants were placed at the same level with the surface of the adjacent cartilage. Wound closure was performed in layers. All rabbits were kept in cages and had free access to food pellets and water. At 6, 12, or 24 weeks after surgery, the rabbits were sacrificed with an intravenous injection of 5 mL pentobarbital (n = 5 rabbits [10 knees] in each group at each time point). Histological Assessment and Staining of Repair Tissue. After sacrifice, the distal portions of the femurs (6 samples in 3 rabbits in each group) were cut off and then fixed in 4% paraformaldehyde (pH 7.4) for 48 hours at 4°C. The femurs were decalcified in 20% EDTA (pH 7.2) in PBS with 5% paraformaldehyde at 4°C. The decalcified specimens were trimmed, dehydrated in a graded ethanol series, and embedded in paraffin. Serial sections (8 mm thick) were cut sagittally through the center of the operative site and stained with hematoxylin and eosin and toluidine blue. Immunohistochemistry was performed with type II collagen antibody (Abcam, Cambridge, UK). Histological Scoring System. A modified method36 was used to evaluate the histological repair of articular cartilage defects. The parameters include 7 categories: cell structure (hyaline cartilage), metachromatic staining of the cartilage matrix, structural integrity of the regenerated cartilage, surface regularity of the tissue, thickness of the cartilage layer, regeneration of the subchondral bone, and integration of the tissue with adjacent cartilage. The scores ranged from 0 to 18 points (see the online Appendix). Higher scores indicate better repairs. Three blinded reviewers were used to evaluate the scores. Scanning Electron Microscopy. The repair tissues were harvested 24 weeks from the surface of the implant in the trochlear groove of the right distal femur. The samples were fixed immediately in a mixture of 4 mL 25% glutaraldehyde and 96 mL 10 mmol/L Tris-HCl (pH 7.4), 4°C for 1 day, then dehydrated with a graded series of 70%, 80%, 90%, and 100% ethanol. Critical point drying was performed in liquid CO2 at 37°C. The specimens were vacuum-coated with a 5-nm layer of gold in a high-vacuum gold spatter coater and then viewed with a scanning electron microscope (S-2500; Hitachi High-Technologies Co, Hitachi-Naka City, Japan). Nanoindentation Assessment of Repair Tissue. The biomechanical analysis of the repair tissues was performed using the nanoindentation based on the reported methods.15,16,23 The samples (n = 9 each group) were isolated from the central part of the repair tissues. The normal hyaline cartilage samples (n = 9) were isolated from the nonoperated trochlear parts of the knee. The circumfluent PBS is to maintain hydration of the samples at room temperature during testing. All indentations were performed using the TriboIndenter (Hysitron Inc, Minneapolis, Minnesota, USA) with a 100-mm radius of a curvature conospherical diamond probe tip. For each indentation, the maximum indentation depth was set to 1000 nm, and the sample was loaded for the first 10 seconds once the tips contacted the
Downloaded from ajs.sagepub.com at DALHOUSIE UNIV on December 26, 2014
586
Dai et al
The American Journal of Sports Medicine
Figure 1. The proliferation and the matrix production of mesenchymal stem cells (MSCs) seeded on the decalcified cortical-cancellous bone matrix (DCCBM) scaffolds. (A) The DCCBM scaffold. (B) The proliferation of MSCs was quantitatively analyzed based on the ratio of total DNA/wet weight of the scaffold. (C and D) The matrix production of MSCs seeded on DCCBM is quantified by the ratio of sulfated glycosaminoglycan (sGAG)/wet weight of scaffold (C) and the ratio of sGAG/DNA content (D) at 1, 4, or 7 days. The MSCs continued to proliferate and produce sGAG with time. Data represent mean 6 standard deviation (n = 6; *P \ .05 vs 1 day). surface of the samples, held at the maximum depth for 2 seconds, and unloaded in the last 10 seconds. The data analysis of nanoindentation was performed based on the reported method.29,30,33 Details are in the online Appendix.
Statistical Analysis For statistical analysis, the statistical significance of the differences between groups was calculated using the KruskalWallis test of analysis of variance (ANOVA). The results from the same group were evaluated using a Student t test. P \ .05 was considered to indicate statistical significance. All results are expressed as mean 6 standard deviation.
RESULTS In Vitro Isolation and Assessment of MSCs Proliferation and Cartilage Matrix Production of MSCs Grown on the DCCBM Scaffold. The DCCBM scaffold used is shown in Figure 1A. We found that both DNA and sGAG content were increased with time (day 7 vs day 1: P = .0001 for DNA, P = .003 for sGAG) (Figure 1, B and C), indicating the MSCs were proliferated from day 1 to day 7 and continued to produce sGAG (cartilaginous matrix) during the
Figure 2. The attachment of mesenchymal stem cells (MSCs) on different parts of the decalcified corticalcancellous bone matrix (DCCBM) scaffold. Scanning electron microscopy shows the growth of MSCs on the surface of cortical parts (A-D) and cancellous parts (E-H) of the DCCBM at different magnifications (original magnification: A and E: 3200, B and F: 3500, C and G: 31000, D and H: 32000). Arrows indicate the typical cells grown on different parts of the scaffold. Confocal laser microscopic images show the growth of MSCs on the cortical parts of the DCCBM (I-L) and the cancellous parts (M-P). The MSCs grown on both parts of the scaffold were stained with green and red fluorescence uniformly (no apoptotic or necrotic cells). Green: the intercalated DNA by acridine orange with fluorescence at 515 to 545 nm. Red: the electrostatic RNA by acridine orange with fluorescence at 590 to 620 nm. Yellow: merged from I and J or M and N. Magnification: 360.
7 days of culture. Also, sGAG/DNA was significantly increased on day 7 compared with day 1 (P = .003) (Figure 1D), indicating that the ability of MSCs to produce sGAG, helpful for cartilage repair, increased after 7 days of culture. Attachment of MSCs Grown on the DCCBM Scaffold. The MSCs were well grown on both parts of the DCCBM scaffold according to images from the scanning electron microscope (SEM; JSM-5600LV; JEOL, Tokyo, Japan). The MSCs spread well (Figure 2, D and H) and adhered tightly to the cortical parts (Figure 2, A-D) and cancellous parts (Figure 2, E-H) of the scaffold, especially in the concave area of the DCCBM scaffold (Figure 2, B and F). Viability of MSCs Grown on the DCCBM Scaffold. A vital dye, acridine orange, was used to assess the viability of MSCs grown on the different parts of the DCCBM. Acridine orange interacts with DNA (515-545 nm) and RNA (590-620 nm). It can distinguish normal, apoptotic, and
Downloaded from ajs.sagepub.com at DALHOUSIE UNIV on December 26, 2014
Vol. 42, No. 3, 2014
One-Step Repair Technique for Cartilage Defects
587
Figure 3. Macroscopic appearance of the cartilage defect healing in 3 groups at 6 (A-C), 12 (D-F), and 24 (G-I) weeks after the operation. The 3 groups are as follows: M group, cartilage defects were treated with the microfracture technique alone; DCCBM group, cartilage defects were implanted with decalcified cortical-cancellous bone matrix (DCCBM) scaffold alone; and DCCBM1M group, cartilage defects were treated with microfracture and implanted with the DCCBM scaffold. necrotic cells. In normal cells, the nucleus was stained green or yellow-green; in apoptotic cells, the nucleus was densely stained with yellow-green fluorescence or yellow-green debris particles; in necrotic cells, the yellow fluorescence weakened or even disappeared. In the current study, the nuclei of MSCs grown on both cortical and cancellous parts were stained with green fluorescence uniformly (Figure 2, I and M). The MSCs were stained with red fluorescence uniformly, indicating that the cytoplasm of the MSCs was uniform, with no rupture and thus no apoptotic cells (Figure 2, J and N). The orientation and the shape of the cells were different. The MSCs in the cortical part appeared as fibroblastic shapes; however, in the cancellous part, they emerged as polygonal shapes, which may have resulted because the cell growth followed the texture of the scaffold. No apoptotic or necrotic cells were seen on these images (Figure 2, I-P).
In Vivo Treatment Groups Macroscopic Findings of the Repaired Cartilage Defects
At 6 Weeks. Repair tissues in the DCCBM1M group had well-filled cartilage defects, many more cells than normal cartilage, better integration, and an irregular surface (Figures 3C and 4C). Small parts (about 2 mm, 50%-60% depth of the defects region) of the cartilage defects were filled
Figure 4. Hematoxylin and eosin staining in 3 groups at 6 (A-C), 12 (D-F), and 24 (G-I) weeks after the operation. (J, K, L) Images of the repair tissues at 24 weeks in each group. (M) Image of the unrepaired cartilage defect with no treatment at 24 weeks. The repair tissues in the DCCBM1M group had a better performance than the other groups. DCCBM, decalcified cortical-cancellous bone matrix; DCCBM1M, perforated DCCBM combined with microfracture; N, normal cartilage; R, repair tissues. The arrows indicate the margins of the normal tissue and the repaired tissue. Black scale bars = 500 mm; white scale bars = 100 mm.
with the repair tissues, with no integration, and clear edges could be seen in the M group and DCCBM group (Figure 3, A and B). In the M group, the repair tissues were thinner and less complete than in the other 2 groups, and the subchondral bone in the defects area was discontinuous (Figure 4A). Occasional cell mass growth could be seen on the scaffold and the edge of normal cartilage in the DCCBM group (Figure 4B). At 12 Weeks. Cartilage defects were well filled with the same repair tissues as that at 6 weeks in the DCCBM1M group. Repair tissues were at the same height of the normal cartilage, and the surface was smoother than at 6 weeks (Figures 3F and 4F). The other 2 groups had improved repair tissues compared with that at 6 weeks, but these tissues were thinner, with no integration
Downloaded from ajs.sagepub.com at DALHOUSIE UNIV on December 26, 2014
588
Dai et al
The American Journal of Sports Medicine
Figure 5. Toluidine blue staining of repaired tissue and normal cartilage in 3 groups at 6 (A-C), 12 (D-F), and 24 (G-I) weeks after the operation. DCCBM, decalcified corticalcancellous bone matrix; DCCBM1M, perforated DCCBM combined with microfracture; N, normal cartilage; R, repair tissues. The arrows indicate the margins of the normal tissue and the repaired tissue. Scale bars = 500 mm. between the regenerated and the normal tissues (Figure 3, D and E). Cracks could be seen between the repaired and the normal tissues in the DCCBM group (Figure 4E). At 24 Weeks. In the DCCBM1M group, repair tissues fully filled in the cartilage defects and had a smooth surface; they were the same as the normal tissue in height, number, and arrangement of the cells (Figures 3I and 4I); moreover, the cell types were hyaline-like chondrocytes (Figure 4L). There was no edge on gross observation (Figure 3I), but little cracks in the hematoxylin and eosin staining (Figure 4I) could be seen between the repair tissues and the normal tissues. The tidemark was clear. However, cartilage defects were smaller than those at 6 and 12 weeks but not fully filled with repair tissues; incomplete integration, an uneven surface, and thinner repair tissues could also be seen in the M group and DCCBM group (Figures 3G, 3H, 4G and 4H); cells were arranged in a haphazard manner (Figure 4, J and K).
Assessment of Cartilage Matrix Production of the Repair Tissues The repair tissues of the M group had no staining with toluidine blue at 6 weeks; the other 2 groups received lighter staining with toluidine blue compared with normal cartilage (Figure 5, A-C). However, lighter staining with toluidine blue and no differences among the 3 groups could be seen at 12 weeks in the repair tissues (Figure 5, D-F). The repair tissues in the DCCBM1M group, which had uniform toluidine blue staining, were much closer to the normal cartilage at 24 weeks (Figure 5I). The other 2 groups had lighter staining compared with normal cartilage at 24 weeks (Figure 5, G and H).
Figure 6. The type II collagen immunohistological staining of repaired tissue and normal cartilage in 3 groups at 6 (A-C), 12 (D-F), and 24 (G-I) weeks after the operation. DCCBM, decalcified cortical-cancellous bone matrix; DCCBM1M, perforated DCCBM combined with microfracture; N, normal cartilage; R, repair tissues. The arrows indicate the margins of the normal tissue and the repaired tissue. Scale bars = 500 mm. Similar to the staining with toluidine blue, staining with collagen type II was especially stronger in the DCCBM1M group compared with the other 2 groups at 6, 12, and 24 weeks after operation and implantation (Figure 6).
Histological Scores The histological scores of the 3 groups are consistent with the results shown above. The score in the DCCBM1M group increased with time (from 10.80 6 2.22 to 12.40 6 2.37, finally increasing to 14.90 6 1.35 at 6, 12, and 24 weeks, respectively). However, little change was seen in the other 2 groups (DCCBM group: from 7.7 6 2.60 to 9.10 6 2.50, finally decreasing to 8.30 6 2.60; M group: from 6.60 6 1.80 to 6.90 6 2.34, finally increasing to 7.30 6 2.19). There were higher scores for repair tissues in the DCCBM1M group compared with the M group (P = .0001) at 12 weeks and compared with both the M group (P = .0001) and DCCBM group (P = .02) at 24 weeks. In addition, the repair tissues in the DCCBM1M group at 24 weeks received higher scores than at 6 weeks (P = .0001) (Figure 7). No such trend was seen in the other 2 groups, indicating that the repaired effects improved with longer duration of implantation.
Assessment of the Integration Between the Normal and Repair Tissues There were cracks between the repair tissues and the normal tissues in the M group and the DCCBM group, although the repair tissues in the DCCBM group were smoother than those in the M group (Figure 8, A and B). However, the edges between the repair tissues and normal
Downloaded from ajs.sagepub.com at DALHOUSIE UNIV on December 26, 2014
Vol. 42, No. 3, 2014
One-Step Repair Technique for Cartilage Defects
589
Figure 7. Histological assessment according to the method described by Wakitani et al36 for the repair tissues in the 3 groups. Higher scores can be seen in DCCBM1M group. DCCBM, decalcified cortical-cancellous bone matrix; DCCBM1M, perforated DCCBM combined with microfracture; M, microfracture alone. *P \ .05.
Figure 8. Scanning electron microscopy images from 3 groups at 24 weeks after the operation. DCCBM, decalcified cortical-cancellous bone matrix; DCCBM1M, perforated DCCBM combined with microfracture; M, microfracture alone; N, normal cartilage; R, repair tissues. A smooth surface can be seen in the repair tissues of the DCCBM1M group. The arrows indicate the margins of the normal and repaired tissue. Scale bars = 500 mm. tissues were not integrated in either the M or the DCCBM group. In the DCCBM1M group, the surface of the repair tissues was smooth, at the same height of the normal cartilage, and well integrated (Figure 8C).
Assessment of the Biomechanical Properties of the Repair Tissues The repair tissues were cut into small round pieces (diameter = 4 mm) for biomechanical analysis (Figure 9A). The typical load-displacement curves in the 3 groups were compared with those in the normal cartilage (Figure 9B). The contact stiffness is considered a suitable measurement for comparing the quality of different repair tissues. Reduced
Figure 9. Mechanical properties of the normal cartilage and the repaired tissue. (A) Samples from the central part of the repaired tissue 24 weeks after transplantation were evaluated by a nanoindenter. (B) Typical load-displacement curves in tissues from 3 groups were compared with the normal cartilage load-displacement curves, providing the tissue response to contact deformation. Contact stiffness (C), reduced modulus (D), and hardness (E) of the repair tissues were compared with the normal cartilage. Higher mechanical properties can be seen in the repair tissues in the DCCBM1M group. DCCBM, decalcified cortical-cancellous bone matrix; DCCBM1M, perforated DCCBM combined with microfracture; M, microfracture alone; N, normal cartilage. (n = 9, *P \ .05) modulus, which represents the elastic deformation that occurs in both sample and indenter tips, is more sensitive to subtle changes in the mechanical behavior. These parameters have been used as key properties in the assessment of cartilage mechanical properties by nanoindentation.15,16,23 In our study, normal cartilage had the highest contact stiffness, followed, in descending order of stiffness, by the samples from the DCCBM1M, DCCBM, and M groups (Figure 9C). Similar results can be seen in the assessment of reduced modulus and hardness (Figure 9, D and E). Normal cartilage displayed the highest contact stiffness, as well as reduced modulus and hardness, while the repair tissues in the M group had the lowest stiffness among the 3 groups (Figure 9, C-E). However, there were
Downloaded from ajs.sagepub.com at DALHOUSIE UNIV on December 26, 2014
590
Dai et al
The American Journal of Sports Medicine
no statistically significant differences between the DCCBM1M group and the normal cartilage in contact stiffness and reduced modulus (Figure 9, C and D); the other groups had significantly lower results compared with normal cartilage (contact stiffness: normal cartilage vs M group, P \ .001, and normal cartilage vs DCCBM group, P \ .001; reduced modulus: normal cartilage vs M group, P \ .001, and normal cartilage vs DCCBM group, P \ .001). These findings may indicate that the biomechanical properties of repaired tissue in the DCCBM1M group were close to those of normal cartilage.
DISCUSSION Recently, single-step repair methods for cartilage repair have been developed to avoid cell manipulation and multiple surgical procedures.11,17 To achieve 1-step cartilage repair, obtaining the cell source is the primary task that needs to be solved. The microfracture technique is a simple method to solve this problem. It can induce MSCs and the chondroprogenitors to migrate into the cartilage lesion and can enhance cytokine release that promotes cartilage repair.24 This method is commonly used in clinics for articular cartilage repairs.22 However, as already reported,26 the cartilage defects usually lead to a fibrocartilage repair. This is probably due to the limited number of cells from the bone marrow, thus making it difficult to induce hyaline cartilage formation.35 However, the surgical approach of creating a microfracture to induce endogenous stem cells seems more favorable than exogenous stem cells in the repair strategy. Also, microfracture can relieve pain, is easy to perform, and can save money for the patient. Therefore, many scaffolds were designed to create a suitable microenvironment to induce MSCs and progenitor cells to acquire chondrocyte-like properties. Previously, native hyaline-like articular cartilage repair was successfully induced by combining microfracture, DCBM, and Ad-BMP4.39 However, the process of the decalcification of DCBM takes too much time because the cortical bone matrix is not porous enough. The use of DCCBM successfully solved this problem. The DCCBM scaffold is more porous (see Appendix Figure A2) and has natural ingredients appropriate for cell growth and the induction of cartilage. The use of a viral vector is always a concern for clinical application.9,38 In the present study, we demonstrated that without the use of Ad-BMP4, the optimized scaffold DCCBM combined with microfracture could also induce a hyalinelike articular cartilage repair. This method is simpler and can avoid the shortcomings of using gene therapy. Moreover, we found that a hyaline-like articular cartilage repair could be observed only in the DCCBM1M group. In the DCCBM-alone group, although the DCCBM scaffold could fill the cartilage defects and the cells could grow on the scaffold, the repair tissues did not appear to have a smooth surface; the matrix staining and the biomechanical properties of repair tissues were weaker than the normal cartilage and the tissues in the DCCBM1M group. In the M group, similar to the other reported studies, the repair tissues were more like a fibrous scar rather than like cartilage.
There are studies that have focused on using MSCs and scaffolds for cartilage repair.8,27,31 Among them, autologous or allogeneic MSCs, growth factors (such as transforming growth factors and bone morphogenetic proteins), and scaffolds are the necessary components, and some require 2 operations to get a thorough regeneration of cartilage. However, our technique is a cell-free, 1-step procedure to get a better regeneration of cartilage. Several factors may contribute to the better repair results. First, the cells induced by the microfracture can migrate to the cartilage defect region. Second, the porous structure of the DCCBM scaffold is conducive to increased cell growth. This is also one of the reasons why the DCCBM scaffold is better than the DCBM scaffold. Third, the DCCBM scaffold itself contains many proteins that may be helpful for the proliferation and differentiation of cells. And fourth, the scaffold promoted cellular production of sGAG, one of the components of cartilage matrix in vitro. Recently, several studies have focused on the 1-step procedure for cartilage repair using biomaterials, such as poly(lactic-co-glycolic) acid (PLGA),12 nanomaterials,21 and hydrogel.20 These are all synthetic materials, and some have not been approved for clinical use. The DCCBM scaffold is made of decalcified bone matrix, a product of processed allograft bone. This is a natural material that has good biocompatibility with the human body and contains proteins that may be helpful for cells compared with other biomaterials. It is easier to get from tissue banks or donors, has been widely used worldwide to repair bone defects, and has been approved for clinical application by the United States Food and Drug Administration. The difference is that the scaffold used in repairing bone defects has not been drilled and may not include the cancellous part of the bone. Because of its ability to measure mechanical properties in situ without disrupting the complex microstructure,14 nanoindentation has been used to measure functional mechanical properties of repaired cartilage in a rabbit knee.13,16 In the present study, contact stiffness, reduced modulus, and hardness were used to assess the biomechanical properties of repair tissues via the nanoindentation technique. However, the indentation modulus is not a constant value in an inhomogeneous tissue such as articular cartilage and is highly dependent on the indentation depth and other factors, such as hydration.16 The modulus values in this study are relative values that aim to compare the different repair tissues under our identical testing conditions. These values may not be simply compared with the results of other reports, because both contact stiffness and reduced modulus are influenced by various methodological and environmental factors.
CONCLUSION The current study offers a promising way to improve 1-step cartilage repair by combining microfracture with the DCCBM scaffold. These findings demonstrate that this biotechnology is easy to perform and leads to hyaline-like cartilage repair in a rabbit model. Long-term studies in other animals as well as in patients are currently in progress.
Downloaded from ajs.sagepub.com at DALHOUSIE UNIV on December 26, 2014
Vol. 42, No. 3, 2014
One-Step Repair Technique for Cartilage Defects
REFERENCES 1. Abedi G, Sotoudeh A, Soleymani M, Shafiee A, Mortazavi P, Aflatoonian MR. A collagen-poly(vinyl alcohol) nanofiber scaffold for cartilage repair [published online December 1, 2010]. J Biomater Sci Polym Ed. 2. Adkisson HD 4th, Martin JA, Amendola RL, et al. The potential of human allogeneic juvenile chondrocytes for restoration of articular cartilage. Am J Sports Med. 2010;38(7):1324-1333. 3. Autologous chondrocytes. Autologous chondrocyte implantation: more data needed. Prescrire Int. 2011;20(116):122-124. 4. Bartlett W, Skinner JA, Gooding CR, et al. Autologous chondrocyte implantation versus matrix-induced autologous chondrocyte implantation for osteochondral defects of the knee: a prospective, randomised study. J Bone Joint Surg Br. 2005;87(5):640-645. 5. Behrens P, Bitter T, Kurz B, Russlies M. Matrix-associated autologous chondrocyte transplantation/implantation (MACT/MACI)—5year follow-up. Knee. 2006;13(3):194-202. 6. Bekkers JE, Inklaar M, Saris DB. Treatment selection in articular cartilage lesions of the knee: a systematic review. Am J Sports Med. 2009;37(suppl 1):148S-155S. 7. Beris AE, Lykissas MG, Kostas-Agnantis I, Manoudis GN. Treatment of full-thickness chondral defects of the knee with autologous chondrocyte implantation: a functional evaluation with long-term followup. Am J Sports Med. 2012;40:562-567. 8. Berninger MT, Wexel G, Rummeny EJ, et al. Treatment of osteochondral defects in the rabbit’s knee joint by implantation of allogeneic mesenchymal stem cells in fibrin clots. J Vis Exp. 2013;(75):e4423. 9. Bouard D, Alazard-Dany D, Cosset FL. Viral vectors: from virology to transgene expression. Br J Pharmacol. 2009;157(2):153-165. 10. Bouwmeester PS, Kuijer R, Homminga GN, Bulstra SK, Geesink RG. A retrospective analysis of two independent prospective cartilage repair studies: autogenous perichondrial grafting versus subchondral drilling 10 years post-surgery. J Orthop Res. 2002;20(2):267-273. 11. Buda R, Vannini F, Cavallo M, Grigolo B, Cenacchi A, Giannini S. Osteochondral lesions of the knee: a new one-step repair technique with bone-marrow-derived cells. J Bone Joint Surg Am. 2010;92(suppl 2):2-11. 12. Chang NJ, Lin CC, Li CF, Wang DA, Issariyaku N, Yeh ML. The combined effects of continuous passive motion treatment and acellular PLGA implants on osteochondral regeneration in the rabbit. Biomaterials. 2012;33(11):3153-3163. 13. Ebenstein DM, Kuo A, Rodrigo JJ, Reddi AH, Ries M, Pruitt L. A nanoindentation technique for functional evaluation of cartilage repair tissue. J Mater Res. 2004;19(1):273-281. 14. Ebenstein DM, Pruitt LA. Nanoindentation of biological materials. Nanotoday. 2006;1(3):26-33. 15. Franke O, Durst K, Maier V, et al. Mechanical properties of hyaline and repair cartilage studied by nanoindentation. Acta Biomater. 2007;3(6):873-881. 16. Gelse K, Muhle C, Franke O, et al. Cell-based resurfacing of large cartilage defects: long-term evaluation of grafts from autologous transgene-activated periosteal cells in a porcine model of osteoarthritis. Arthritis Rheum. 2008;58(2):475-488. 17. Giannini S, Buda R, Vannini F, Cavallo M, Grigolo B. One-step bone marrow–derived cell transplantation in talar osteochondral lesions. Clin Orthop Relat Res. 2009;467(12):3307-3320. 18. Hangody L, Dobos J, Balo E, Panics G, Hangody LR, Berkes I. Clinical experiences with autologous osteochondral mosaicplasty in an athletic population: a 17-year prospective multicenter study. Am J Sports Med. 2010;38(6):1125-1133. 19. Johnson LL. Arthroscopic abrasion arthroplasty: a review. Clin Orthop Relat Res. 2001;391(suppl):S306-S317.
591
20. Kitamura N, Yasuda K, Ogawa M, et al. Induction of spontaneous hyaline cartilage regeneration using a double-network gel: efficacy of a novel therapeutic strategy for an articular cartilage defect. Am J Sports Med. 2011;39(6):1160-1169. 21. Kon E, Delcogliano M, Filardo G, Busacca M, Di Martino A, Marcacci M. Novel nano-composite multilayered biomaterial for osteochondral regeneration: a pilot clinical trial. Am J Sports Med. 2011;39(6):1180-1190. 22. Kreuz PC, Steinwachs MR, Erggelet C, et al. Results after microfracture of full-thickness chondral defects in different compartments in the knee. Osteoarthritis Cartilage. 2006;14(11):1119-1125. 23. Li C, Pruitt LA, King KB. Nanoindentation differentiates tissue-scale functional properties of native articular cartilage. J Biomed Mater Res A. 2006;78(4):729-738. 24. Lotz M. Cytokines in cartilage injury and repair. Clin Orthop Relat Res. 2001;391(suppl):S108-S115. 25. Matsunaga D, Akizuki S, Takizawa T, Yamazaki I, Kuraishi J. Repair of articular cartilage and clinical outcome after osteotomy with microfracture or abrasion arthroplasty for medial gonarthrosis. Knee. 2007;14(6):465-471. 26. Miller BS, Steadman JR, Briggs KK, Rodrigo JJ, Rodkey WG. Patient satisfaction and outcome after microfracture of the degenerative knee. J Knee Surg. 2004;17(1):13-17. 27. Morille M, Van-Thanh T, Garric X, et al. New PLGA-P188-PLGA matrix enhances TGF-beta3 release from pharmacologically active microcarriers and promotes chondrogenesis of mesenchymal stem cells. J Control Release. 2013;170(1):99-110. 28. Moseley JB Jr, Anderson AF, Browne JE, et al. Long-term durability of autologous chondrocyte implantation: a multicenter, observational study in US patients. Am J Sports Med. 2010;38(2):238-246. 29. Oliver WC, Pharr GM. An improved technique for determining hardness and elastic-modulus using load and displacement sensing indentation experiments. J Mater Res. 1992;7(6):1564-1583. 30. Oliver WC, Pharr GM. Measurement of hardness and elastic modulus by instrumented indentation: advances in understanding and refinements to methodology. J Mater Res. 2004;19(1):3-20. 31. Park JS, Woo DG, Yang HN, Lim HJ, Chung HM, Park KH. Heparinbound transforming growth factor-beta3 enhances neocartilage formation by rabbit mesenchymal stem cells. Transplantation. 2008;85(4):589-596. 32. Peterson L, Vasiliadis HS, Brittberg M, Lindahl A. Autologous chondrocyte implantation: a long-term follow-up. Am J Sports Med. 2010;38(6):1117-1124. 33. Pharr GM, Oliver WC, Brotzen FR. On the generality of the relationship between contact stiffness, contact area, and elastic modulus during indentation. J Mater Res. 1992;7(3):613-617. 34. Schindler OS. Current concepts of articular cartilage repair. Acta Orthop Belg. 2011;77(6):709-726. 35. Steadman JR, Briggs KK, Rodrigo JJ, Kocher MS, Gill TJ, Rodkey WG. Outcomes of microfracture for traumatic chondral defects of the knee: average 11-year follow-up. Arthroscopy. 2003;19(5):477484. 36. Wakitani S, Goto T, Pineda SJ, et al. Mesenchymal cell-based repair of large, full-thickness defects of articular cartilage. J Bone Joint Surg Am. 1994;76(4):579-592. 37. Williams RJ 3rd, Harnly HW. Microfracture: indications, technique, and results. Instr Course Lect. 2007;56:419-428. 38. Young LS, Searle PF, Onion D, Mautner V. Viral gene therapy strategies: from basic science to clinical application. J Pathol. 2006;208(2):299-318. 39. Zhang X, Zheng Z, Liu P, et al. The synergistic effects of microfracture, perforated decalcified cortical bone matrix and adenovirusbone morphogenetic protein-4 in cartilage defect repair. Biomaterials. 2008;29(35):4616-4629.
For reprints and permission queries, please visit SAGE’s Web site at http://www.sagepub.com/journalsPermissions.nav
Downloaded from ajs.sagepub.com at DALHOUSIE UNIV on December 26, 2014
