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Gene Therapy (2010) 17, 779–789 & 2010 Macmillan Publishers Limited All rights reserved 0969-7128/10 $32.00 www.nature.com/gt ORIGINAL ARTICLE Articular cartilage repair by genetically modified marrow aspirate in sheep

A Ivkovic1, A Pascher2, D Hudetz3, D Maticic4, M Jelic5, S Dickinson6, M Loparic7,8, M Haspl9, R Windhager2 and M Pecina10 1University Hospital Center Zagreb, School of Medicine, University of Zagreb, Zagreb, Croatia; 2Department of Orthopaedic Surgery, Medical University of Graz, Graz, Austria; 3University Hospital for Traumatology, School of Medicine, University of Zagreb, Zagreb, Croatia; 4Department of Surgery, Orthopaedics and Ophthalmology, Veterinary School, University of Zagreb, Zagreb, Croatia; 5Department of Orthopaedic Surgery, University Hospital Center Zagreb, Zagreb, Croatia; 6Department of Cellular and Molecular Medicine, School of Medical Sciences, University of Bristol, Bristol, UK; 7Departments of Surgery and Biomedicine, University Hospital Basel, Basel, Switzerland; 8ME Mueller Institute for Structural Biology, Biozentrum University of Basel, Basel, Switzerland; 9Special Hospital for Orthopaedic Surgery ‘Akromion’, Krapinske Toplice, Croatia and 10Department of Orthopaedic Surgery, School of Medicine, University of Zagreb, Zagreb, Croatia

Bone marrow presents an attractive option for the treatment and biomechanical parameters. Assessment of repair of articular cartilage defects as it is readily accessible, it showed that the groups treated with constructs transplanta- contains mesenchymal progenitor cells that can undergo tion contained more cartilage-like tissue than untreated chondrogenic differentiation and, once coagulated, it provides controls. Improved cartilage repair was observed in groups a natural scaffold that contains the cells within the defect. This treated with unmodified plugs and Ad.TGF-b1- study was performed to test whether an abbreviated ex vivo transduced plugs, but the repaired tissue from TGF-treated protocol using vector-laden, coagulated bone marrow aspi- defects showed significantly higher amounts of II rates for gene delivery to cartilage defects may be feasible for (Po0.001). The results confirmed that the proposed method clinical application. Ovine autologous bone marrow was is fairly straightforward technique for application in clinical transduced with adenoviral vectors containing cDNA for settings. Genetically modified bone marrow clots are suffi- green fluorescent or transforming growth factor cient to facilitate articular cartilage repair of partial-thickness (TGF)-b1. The marrow was allowed to clot forming a gene defects in vivo. Further studies should focus on selection of plug and implanted into partial-thickness defects created on transgene combinations that promote more natural healing. the medial . At 6 months, the quality of articular Gene Therapy (2010) 17, 779–789; doi:10.1038/gt.2010.16; cartilage repair was evaluated using histological, biochemical published online 11 March 2010

Keywords: adenovirus; articular cartilage defects; transforming growth factor-b1; bone marrow; cell transplantation

Introduction often lead to premature , consequently decreasing quality of life for the patient and adding is a highly specialized tissue, with a substantially to health care costs. unique three-dimensional structure that enables it to The restoration of damaged cartilage remains one of withhold tremendous mechanical forces inflicted during the biggest challenges in modern orthopaedics. There is movement and a smooth surface allowing nearly no pharmacological treatment that promotes the repair of frictionless contact between articulating joint surfaces. the cartilage. Current treatment modalities include However, this tissue is also avascular, aneural and microfracture, transplantation of ostechondral grafts alymphatic with a low cell density embedded within and , use of biodegradable scaffolds or the (ECM). For these reasons, hyaline combination of these.1,2 Although these procedures can cartilage has very modest reparative and regenerative produce initially good clinical results in terms of pain capabilities. Articular cartilage defects are very frequent, relief and improvement of joint function, they typically especially among active young adults and the working produce a fibrocartilagenouse repair tissue that is population. Such defects do not heal and, with time, inferior to normal cartilage, such that long-term out- comes are less predictable and satisfactory.3 New biological approaches to cartilage repair that are Correspondence: Dr A Ivkovic, Clinical Institute for Orthopaedic based on the use of cells and molecules that promote Aids and Rehabilitation, University Hospital Center Zagreb, or/and inhibit cartilage breakdown offer Bozidareviceva 11, Zagreb 10000, Croatia. 4 E-mail: [email protected] a promising alternative to current treatment options. Received 5 July 2009; revised 15 October 2009; accepted 16 Any successful strategy that attempts to repair hyaline November 2009; published online 11 March 2010 cartilage defects must include sufficient number of cells, Gene therapy in ovine articular cartilage repair A Ivkovic et al 780 appropriate signal to modulate cellular response and a Results scaffold that would contain the cells within the defect. Mesenchymal stromal cells (MSCs) present very attrac- Histological assessment tive option for cell-based strategies as they can be readily The mean scores of the histological assessment are isolated, expanded and, under appropriate conditions, shown in Table 1, and representative histological sections differentiated into mesenchymal tissues such as cartilage, are shown in Figure 1. Six months after the surgical bone or muscle.5 Numerous gene products such as procedure, all groups treated with BMC transplantation transforming growth factor-b (TGF-b),6 bone morphoge- were superior to empty control in terms of overall score netic protein-7 (BMP-7),7 insulin-like growth factor-1 according to the ICRS Visual Histological Assessment (IGF-1)8 and BMP-29 have shown potential in regulating Scale, although statistical significance was not observed the process of growth, repair and regeneration of (P ¼ 0.061) (Table 1). Each histological parameter was cartilage in animal models, but their use is limited by analysed by Kruskal–Wallis test. A statistically signifi- delivery problems and rapid clearance from the joint. cant difference was observed in one category—columnar This issue can be addressed by gene therapy approaches cell distribution: TGF and BMC groups had a higher for delivering therapeutic gene products specifically to score than the CON group (Table 1). Although improved the site of repair.10,11 In particular, viral vectors have been fusion between the repair tissue and the surrounding used successfully to modify graftable articular chondro- undamaged tissue had been achieved in TGF-treated cytes, periosteal cells and bone marrow-derived MSCs ex groups, the side-to-side integrations were far from vivo, as well as for directly modifying the synovial lining optimal. in vivo.12,13 The use of scaffolds in cartilage repair is often required to contain, deliver and orient cells within their In vitro culturing of genetically modified BMCs three-dimensional structure. Many different types have BMCs transduced with green fluorescent protein been tested in clinical and experimental settings, but the (Ad.GFP) showed a high number of fluorescent cells selection of suitable scaffolds for clinical application is throughout the coagulate over 21 days of culture. BMCs still ongoing.14 formed in the absence of Ad.GFP confirmed the Much of the current research into cartilage repair has specificity of fluorescence signal. focused on ex vivo, or indirect, approaches to the treatment of defects using genetically modified cells. Biochemical properties These typically involve harvesting and expansion of the (GAG) analysis did not reveal any cells, transduction with a therapeutic gene, seeding onto statistical difference between mean values for repaired a scaffold and reimplantation into the defect. While cartilage in the treatment groups and native cartilage providing the aforementioned components for successful from contralateral (P40.050 for all comparisons, cartilage repair, this general approach is technically paired samples t-test; Figure 2a). There were no demanding, expensive and requires at least two surgical statistically significant differences in GAG mean values 15 procedures. Pascher et al. have recently proposed an for repaired cartilage between the treatment groups abbreviated ex vivo protocol that uses vector-laden, (F2,19 ¼ 0.6, P ¼ 0.581, one-way analysis of variance coagulated bone marrow aspirates or ‘gene plugs’, for (ANOVA); Figure 2a). gene delivery to cartilage defects. This study showed that nucleated cells within fresh autologous bone marrow aspirates may be successfully transduced with adeno- Table 1 Histological grading of the repair tissue at 6 months viral vectors sufficient to secret transgene products up to according to ICRS visual histological assessment Scalea (median- 21 days. In theory, this approach also provides all s±interquartile range) necessary ingredients for successful cartilage repair: transduced mononuclear cells secrete signals that stimu- ICRS Score Treatment group late mesenchymal progenitors to differentiate along the chondrogenic lineage, and the bone marrow clot (BMC) CON BMC GFP TGF Pb itself provides a natural autologous three-dimensional scaffold to be used for containment of cells and vectors CQCQCQCQ within the defect. To examine whether direct implantation of gene plugs Surface 0.00 0 0.00 0 0.00 0 0.00 0 0.343 might be used in situations that closely mimic real-life Matrix 1.50 2 3.00 1 3.00 2 3.00 0 0.062 clinical situations, partial-thickness chondral defects Columnar cell distribution 1.00c 0 2.00 0 2.00 2 2.00 0 0.016 were created on the weight-bearing surface of the Cell population viability 0.00 1 1.00 3 0.00 0 0.00 0 0.095 Subchondral bone 2.50 1 3.00 0 3.00 3 3.00 0 0.177 femoral condyle in sheep. Fresh autologous bone Cartilage mineralization 1.50 3 3.00 0 3.00 3 3.00 0 0.162 marrow aspirates were transduced with adenoviral Median total score 6.50 6 11.50 4 11.00 9 11.00 0 0.061 constructs carrying therapeutic or marker genes, and the clots were press-fit within the defects. The three Abbreviations: CON, control group; BMC, bone marrow clot group; principle objectives of this study were to determine GFP, green fluorescent protein group; TFG, transforming growth feasibility of the proposed abbreviated ex vivo protocol to factor-b1 group, C, median; Q, interquartile range. be used as a novel treatment tool in clinical settings, to aThe table shows the medians from each group for each b subcategories, and the total medians for each group. determine whether transgene expression of TGF- 1 b within the gene plug enhances cartilage repair, and to Kruskal–Wallis test (Mann–Whitney test was used as a post hoc procedure when K–W test revealed statistically significant differ- test whether there is a presence of adenoviral genome ence). within the cells of synovial lining, which would suggest cSignificantly lower score from TGF and BMC groups (P ¼ 0.002 and vector leakage from the clots. P ¼ 0.008, respectively, Mann–Whitney test).

Gene Therapy Gene therapy in ovine articular cartilage repair A Ivkovic et al 781

Figure 1 Representative histological sections of the repair tissue filling ovine chondral defects, stained with either haematoxylin-eosin (upper row) or safranin-O (lower row). Panel bars: 100 mm. (a, b) Empty defect group showing acellular tissue (Ac) within the defect with intact calcified layer (Ca) and subchondral bone (B). (c, d) BMC group. The defect is predominantly filled with fibrocartilage (Hf). There is clear demarcation between native hyaline cartilage (Hc) and fibrocartilage (Hf) separated by a defect gap (DG). Black arrow indicates border of the defect separating newly formed (New) and native (Nat) cartilage. Calcified layer (Ca). (e, f) GFP group. Irregular filling of the defect with fissures. The defect is filled with mixture of hyaline and fibrocartilage. Clusters of clonal cell division (Cl) are present in upper layers newly formed tissue. Black arrow indicates border of the defect separating newly formed (New) and native (Nat) cartilage. Calcified layer (Ca). (g, h) TGF group. Improved histological appearance of the repair tissue within the defect. Hyaline cartilage (Hc) and columnar organization of chondrocytes is detected on both sides of the defect gap (DG). Black arrow indicates border of the defect separating newly formed (New) and native (Nat) cartilage. Calcified layer (Ca).

25 10 † 9 † 20 8 † 7 15 6 Repaired 5 Repaired Cartilage 10 Cartilage 4 dry weight) 3 Native Cartilage 5 Native 2 Cartilage Collagen I (as % of digested 1 * * * 0 0 GAG (as % of digested dry weight) BMC GFP TGF BMC GFP TGF

† 90 90 † † 80 80 70 70 * * 60 * 60 50 * 50 Repaired Repaired 40 Cartilage 40 Cartilage Water (%) dry weight) 30 Native 30 Native Cartilage 20 Cartilage 20 10 10 Collagen II (as % of digested 0 0 BMC GFP TGF BMC GFP TGF Figure 2 Biochemical analysis of repaired cartilage compared with native cartilage. (a) GAG. (b) Collagen I. (c) Collagen II. (d) Water. *Two- tailed paired t-test: comparison of repaired and native cartilage (Po0.05). wOne-way ANOVA with Tukey post hoc test: comparison of treatment groups (Po0.05).

The collagen type I content was found to be isons, paired samples t-test; Figure 1b). The three significantly higher in all treatment groups when treatment groups also significantly differed in collagen compared with native cartilage (Po0.050 for all compar- type I content (F2,19 ¼ 13.9, Po0.001, one-way ANOVA;

Gene Therapy Gene therapy in ovine articular cartilage repair A Ivkovic et al 782 Figure 2b). Specifically, the collagen type I content in 3 BMC group was significantly lower from that detected in GFP- and TGF-treated groups, respectively (Po0.001 2.5 † and P ¼ 0.001, respectively, Tukey post hoc test), whereas there was no difference among GFP and TGF groups 2 (P ¼ 0.482, Tukey post hoc test).

(MPa) Repaired 1.5 The collagen type II content was significantly lower in Cartilage * BMC and GFP treatment groups when compared with micro * * Native

*| 1 native cartilage (Figure 2c). There was no difference Cartilage E between the GFP and BMC groups (P ¼ 0.079, Tukey post | hoc test). Collagen type II content in the TGF group 0.5 Dynamic elastic modulus was significantly higher (F2,19 ¼ 56.2, Po0.001, one-way ANOVA; Figure 2c) than that detected in GFP- and BMC- 0 BMC GFP TGF treated groups (Po0.001 for both, Tukey post hoc test). When compared with native cartilage, water content † in the repaired tissue was significantly lower in the TGF 300 †† and GFP groups (Po0.001 and P ¼ 0.005, respectively, 250 paired samples t-test; Figure 2d), whereas the water content of the TGF group was significantly lower than 200 those detected in the GFP (F2,19 ¼ 5.9, P ¼ 0.01, one-way 150 ANOVA; Figure 1d) and BMC groups (P ¼ 0.008, Tukey Repaired (KPa) post hoc test). 100 Cartilage

nano Native

*| 50

E * Cartilage

| * Biomechanical properties 0 Cartilage stiffness at micrometer scale. BMC GFP TGF Dynamic Dynamic elastic modulus -50 elastic modulus |E*| values for native articular cartilage and reparative cartilage obtained after treatment with -100 gene plugs are shown in Figure 2. The measurements Figure 3 (a) Micrometer measurements. Dynamic elastic modulus were obtained with a microspherical tip, nominal radius |E*|micro of native articular cartilage and repair tissue of the BMC, of 7.5 mm, and they reflect structural changes at the GFP and TGF group. Average microstiffness (M±s.d.) increased ± micrometer scale. |E*|micro values gradually increased from native cartilage to GFP-treated group: |E*|micro ¼ 1.025 0.098 ± from native cartilage to the TFG-, BMC- and GFP-treated (native cartilage), |E*|micro ¼ 1.577 0.285 (TGF), |E*|micro ¼ ± ± groups (Figure 3a). |E*| was significantly higher in 1.863 0.079 (BMC), |E*|micro ¼ 2.025 0.371 (GFP). *Two-tailed micro paired t-test comparison of repaired versus native cartilage all repair groups when compared with native cartilage (Po0.05). wOne-way ANOVA with Tukey post hoc test comparison (BMC Po0.001, GFP P ¼ 0.003, TGF Po0.001, respec- of repaired cartilage between treatment groups (Po0.05). (b) Nano-

tively, paired samples t-test). Treatment groups signifi- meter measurements. Dynamic elastic modulus |E*|nano of native cantly differed in |E*|micro (F2,19 ¼ 5.3, P ¼ 0.015; one- articular cartilage and repair tissue of the BMC, GFP and TGF ± ± way ANOVA). |E*| from the TGF group was group. |E*|nano ¼ 19.28 3 (native cartilage), |E*|nano ¼ 21.54 1.24 micro ± ± significantly lower than that detected in the GFP group (BMC), |E*|nano ¼ 39.26 104.56 (TGF), |E*|nano ¼ 189.21 39.26 (GFP). *Two-tailed paired t-test comparison of repaired and native ¼ w (P 0.014, Tukey post hoc test). Values from the TGF cartilage (Po0.05). One-way ANOVA with Tukey post hoc test group also appeared lower compared with those of the comparison of treatment groups (Po0.05). BMC group, but no statistically significant difference was observed. Table 2 Associations between dynamic elastic modulus measured There was a moderately positive association of on micrometer and nanometer scale and biochemical parameters |E*|micro with water and a moderately negative associa- (GAG, collagen I, collagen II, and water) tion with collagen type II, but no correlation with GAG and collagen type I content (Table 2). Association [Spearman’s r(p)]

|E*| |E*| Cartilage stiffness at nanometer scale. Dynamic micro nano elastic modulus |E*| values for native articular cartilage GAG À0.35 (0.108) À0.24 (0.288) and reparative cartilage are presented in Figure 3b. The Collagen I À0.08 (0.710) 0.80 (o0.001)a measurements were obtained with sharp pyramidal tip, Collagen II À0.56 (0.007)a 0.29 (0.191) nominal radius of 20 nm, and they reflect structural Water 0.44 (0.038)b À0.46 (0.033)b changes at the nanometer scale. Obtained |E*|nano values suggested that the BMC-treated group had very Abbreviation: GAG, glycosaminoglycan. similar stiffness to native cartilage (P ¼ 0.345, paired aAssociation is significant at the 0.01 level (two-tailed). samples t-test), but that stiffness was higher in TGF- and bAssociation is significant at the 0.05 level (two-tailed). GFP-treated groups (P ¼ 0.028 and P ¼ 0.005, respec- tively, paired samples t-test; Figure 3b). Furthermore, compared with both GFP and TGF (P ¼ 0.001 and

statistically significant differences in |E*|nano were P ¼ 0.004, respectively, Mann–Whitney test). found between treatment groups (Po0.001, Kruskall– There was a strong positive association of |E*|nano Wallis test). Stiffness was significantly higher in the GFP with collagen I content and a moderate negative control group than in the TGF group (P ¼ 0.007, Mann– association with water content, but no correlation with

Whitney test), whereas BMC had lower |E*|nano when GAG and collagen II content (Table 2).

Gene Therapy Gene therapy in ovine articular cartilage repair A Ivkovic et al 783 Polymerase chain reaction analysis step operation, which can be easily done by two To determine any expression levels from the gene surgeons within 30–45 min. transfer vectors within the 6 months after surgery, polymerase chain reaction (PCR) was performed using primer sets recognizing the cytomega- The use of TGF-b1-transduced BMCs for articular lovirus (CMV) promoter as well as sheep b-actin. The cartilage defects repair analysis included five groups of specimens according to Adult MSCs present a very interesting platform for the treatment, namely TGF-b1 vector-treated, GFP vector- development of treatment strategies in orthopaedic treated as a transduction control, bone marrow treated, . They can be obtained relatively easily empty defect group and controls from contralateral . from various tissue sources such as bone marrow, fat and PCR analysis of the synovial tissue revealed no presence muscle, and under appropriate conditions they have the of the CMV promoter in any of the treatment groups or capacity of differentiation into various mesenchymal the control group 180 days after implantation. Expres- lineages including bone and cartilage.5,26 Numerous in sion of the b-actin gene was detected in all of the vitro studies showed that primary MSCs undergo analysed samples. chondrogenic differentiation when cultured in the pre- sence of specific media supplements, including dexa- methasone and certain extracellular biological cues.27,28 Discussion TGF-b1 has been used as a key inductor of chondro- genesis in many in vivo and in vitro studies, as it It is well known that the healing of focal lesions in adult stimulates cell proliferation and synthesis of major articular cartilage is very limited and, over time, they components of ECM, GAG and collagen.29–31 It was may progress to osteoarthritis. Articular cartilage da- chosen for use in this study because it is one of the best mage is a growing health care problem and a recent characterized and most potent chondrogenic growth study showed that approximately two thirds of patients factors. The results of this study showed that all groups undergoing knee have been diagnosed that underwent transplantation of BMCs have a high with cartilage lesions.23 On the other hand, the growing content of GAGs, but only the repair tissue from defects armamentarium of novel biological methods and tech- treated with TGF-b1 gene plugs had a very high content nologies offer scientists and clinicians powerful tools for of collagen type II similar to native cartilage. The fact that developing effective new methods in treating damaged only TGF-treated defects scored statistically higher in cartilage. The cornerstones on which a successful tissue terms of columnar cellular distribution suggests that engineering-based strategy will likely be built include an residing mesenchymal progenitors within the gene plug adequate supply of repair cells, sufficient induction of responded to the local expression of TGF-b1 in terms of those cells by growth factor signalling, and a biocompa- chondrogenic differentiation, which ultimately resulted tible scaffold conducive to the repair process.24 in higher ECM turnover and better quality of the The approach to focal cartilage defect repair described cartilage repair. Guo et al.32 reported similar results in a in this study uses vector-laden coagulated bone marrow rabbit model of full-thickness cartilage defects using an aspirates for gene delivery to the defect site. Aspirated ex vivo approach and a chitosan scaffold. Another study autologous bone marrow contains progenitor cells, the by Pagnotto et al.33 showed improved cartilage repair in matrix is completely natural and native to the host, and osteochondral defects implanted with MSCs transduced the constitutive fibrin fibres adhere the whole construct with adeno-associated virus (AAV) carrying cDNA for to the surface of the defect. Preliminary in vitro and in TGF-b1. In their study, transgene expression slowly vivo studies on small animals showed that clotted decreased from 100% at 2 weeks to 17% at 12 weeks, mixtures of adenoviral suspensions with fresh aspirated but it proved that gene therapy enables sustained bone marrow resulted in levels of transgenic expression delivery of the bioactive molecules for a period of time in direct proportion to the density of nucleated cells that is sufficient to induce and govern cellular response within the clot.15 This study is a step forward towards a within the defect. Owing to its safe profile, AAV is clinical application of these gene plugs to treat local considered to be the most suitable viral vector for human cartilage lesions. The whole study was conceived in a application, and is currently being tested in a phase I way to simulate potential clinical situation where one clinical trial.34 would have to treat isolated chondral defect situated on Integration of newly formed cartilage with and the load-bearing surface of the femoral condyle. There- neighbouring, undamaged tissue has always been a fore, sheep were chosen for a large animal model. One major issue in cartilage repair. Poor integration causes drawback of the proposed model is the fact that sheep uneven distribution of mechanical loading, and predis- cartilage on the medial condyle is very thin. Ahern et al.25 poses the joint surface to the development of early performed a detailed systematic review of preclinical degenerative changes. Although certain improvement in animal models in single-site cartilage defect testing. integration was observed in TGF-treated groups, the According to their analysis, the ovine cartilage is variable overall integration is far from being optimal. in thickness and it measures from 0.40–1.68 mm. Minor Considering that a number of biological factors act in a variability in the obtained results might be contributed to highly coordinated manner during native tissue devel- that fact, nevertheless, reproducible standardized chon- opment, the use of a single factor to stimulate and dral defects could be created in all animals, using an regulate process of chondrogenic differentiation, while adapted punch-drill device. For implantation of the gene practical, has limitations with regard to the producing plugs, standard operation instruments were used. The cartilage of optimal quality. Chondrogenesis is a finely proposed method proved to be a fairly straightforward regulated process, which includes numerous growth and technique for application in clinical settings. It is a single- transcription factors, and a combination of these might

Gene Therapy Gene therapy in ovine articular cartilage repair A Ivkovic et al 784 be more effective. For example, synergistic effects on network of different types of collagen fibrils. Under chondrogenesis have been reported for TGF-b1 when cyclic loading, the applied stress and resulting strain are co-administered with IGF-1.35 Steinert et al.36 recently not in phase. To determine stiffness of the cartilage, used an aggregate culture system to study effects of compressive force is applied and the ratio of stress to co-expression of TGF-b1, IGF-1 and BMP-2 on MSCs. strain, the dynamic elastic modulus |E*|, is calculated. Their results showed larger aggregates, higher levels Several studies describe the use of probes of varying of GAG synthesis and greater expression of cartilage shape for indentation testing of cartilage where data specific marker genes by adding different combinations are typically assessed at millimeter scale. However, of growth factors. Furthermore, it is known that TGF this is insufficient to detect local mechanical stimulation of MSCs promotes hypertrophy and the property variations of the examined tissue that reflect increased expression of collagen type I and X. However, differences in cartilage structural organization at the Kafienah et al.37 have shown that including parathyroid molecular level.41,42 hormone-related protein downregulates collagen type I To overcome these limitations, Stolz et al.22 proposed a and X in cartilage tissue engineered from MSCs. It novel, AFM-based approach they termed IT AFM. Their should be also noted that some transcription factors such protocol enabled absolute measurements of the dynamic as Sox-9 (which is known to be essential for the full elastic modulus |E*| at two different length scales of expression of phenotype) and Wnt are not tissue organization—micrometer and nanometer. This is chondrogenic itself, but can make cells more responsive technically possible because two different probe types to growth factors and other chondrogenic stimuli. Along are used for these measurements: the microspherical tips these lines, to optimize the proposed method, delivery of for micrometerscale measurements, and sharp pyramidal multiple genes might be more reliable option, and tips for nanometerscale measurements. In our study,

further studies are needed to pinpoint the exact protocol dynamic elastic modulus |E*|nano of the native sheep B B in terms of concentration and temporal sequence of cartilage is 0.02 MPa, and |E*|micro is 1 MPa, which delivery of chosen genes. is in agreement with studies performed on human Another important drawback to our study was the fact cartilage, where stiffness values averages around 0.015 that we were not able to control weight-loading condi- and 2.6 MPa, respectively, for healthy individuals tions in the operated animals. Inconsistencies in repair without OA.43,44 According to Stolz et al., this 100-fold quality within the treatment groups could be attributed modulus difference between micrometer and nanometer to the influence of the weight-loading conditions of the scale is a result of assessing different levels of cartilage joint immediately after the surgical procedure. In human hierarchical organization. On the micrometer scale, patients, proper rehabilitation protocols are crucial to articular cartilage behaves as relatively amorphous optimize the results of bone marrow stimulating as well material, whereas at the nanometer scale, ultrastructural as cell-based techniques, including postoperative con- differences are resolved. tinuous passive motion exercises along with crutch- Microstiffness values were lowest for native cartilage assisted restrictions of weight-bearing up to 6–8 and gradually rose from TGF- and BMC- to GFP-treated weeks.38,39 Practical limitations prevented postoperative groups, respectively (Figure 2). This would suggest that ambulation restrictions, possibly allowing detrimental the repair tissue of the TGF-treated group is qualitatively shear forces on the construct and leading to a reduced superior to the other two groups showing biomechanical quality of produced matrix. These limitations might be properties close to native cartilage. However, nanoscale reflected in the fact that TGF-treated groups had good measurement showed that the BMC-treated group has concentrations of GAGs and collagen II, but very high very similar nanostiffness to that of native cartilage, and content of collagen I and low content of water. the stiffness values of the TGF- and GFP-treated groups are much higher (Figure 3). We hypothesized that this observation could reflect different amounts and spatial Determining biomechanical properties of cartilage orientation of newly synthesized extracellular compo- repair tissues by indentation-type atomic force nents and/or water content within the repair tissue in microscopy the last two groups. To test this hypothesis, correlation Biochemical and histological parameters provide infor- analysis was performed, which showed that the dynamic

mation regarding the amount and spatial distribution of elastic modulus |E*|micro correlated moderately posi- the major components comprising repaired cartilage. tively with water and moderately negatively with However, only biomechanical analysis can assess the collagen type II, but not with GAG and collagen type I

load-bearing capabilities of the cartilage and therefore content (Table 2). At the same time, |E*|nano correlates biomechanical parameters reflect the functionality of the strongly positive with collagen type I and moderately repaired tissue. To determine load-bearing capabilities of negative with water, but not with GAG and collagen type examined tissue, indentation-type atomic force micro- II content (Table 2). At the micrometer level, biomecha- scopy (IT AFM) was used to determine stiffness—a nical properties of cartilage repair tissue are only mechanical parameter that describes the relation between moderately correlated with the biochemical content. This an applied, nondestructive load and resultant viscoelastic observation leads us to conclusion that, at micrometer deformation of cartilage tissue. Furthermore, biomecha- level of tissue organization, it is not possible to determine nical data with biochemical content was correlated. contribution of individual ECM components to bio- Hyaline cartilage is highly specialized tissue with mechanical properties of repaired cartilage. However, unique three-dimensional structure, which allows it at the nanometer level, dynamic elastic modulus to behave mechanically as a viscoelastic solid.40 This correlates with collagen I content, which is barely present reflects the unique ultrastructure of cartilage ECM, in native cartilage. A sharp AFM tip has nominal radius which is composed of embedded into a of 20 nm that is smaller than an individual collagen fibril,

Gene Therapy Gene therapy in ovine articular cartilage repair A Ivkovic et al 785 which typically measures around 50 nm.22 Although overexpression of TGF-b1 (Ad.TGF-b1) and Ad.GFP— microspherical tip is too big to detect subtle differences each driven by the CMV promoter—were constructed by in orientation and amount of collagen fibrils, sharp Cre-lox recombination using the system of Hardy et al.17 pyramidal tip can discriminate such differences, result- These adenoviral vectors were propagated in 293-CRE8 ing in higher stiffness values. cells and purified on three successive CsCl2 density gradients between 1.2 and 1.4 g mlÀ1. Following dialysis

Presence of adenoviral vector in the surrounding in 10 mM Tris-HCL, pH 7.8, 150 mM NaCl, 10 mM MgCl2 synovial lining and 4% sucrose, the preparations were aliquotted and The use of viral-based gene therapy is controversial with stored at À80 1C. Viral titers were estimated by optical regards to safety. Although very effective in terms of density and standard plaque assay. gene transfer and expression, viral vectors induce immune response and their presence in the surrounding Animals tissue may result with detrimental side effects. For Twenty-eight skeletally mature sheep (female, 1–3 years example, TGF-b1 administered into the joint in sufficient old) were used for this study. The sheep were randomly concentrations can lead to chondrophyte formation at the assigned to one of the four groups. In the BMC group joint margins, which at later stages calcify and become (n ¼ 6), the sheep were implanted with untreated auto- real osteophytes.45 One of the major goals of this study logous BMC that was aspirated from iliac crest of was to determine whether there is any residual presence respective animal. In the GFP group (n ¼ 6), autologous of virus within the synovium. After killing the animals, BMCs genetically modified with Ad.GFP were implanted were inspected for any signs of osteophyte in sheep as a vector control. In the TGF-b1-treated (TGF) formation and/or arthrofibrosis, but none was detected. group (n ¼ 10), autologous BMCs genetically modified Although PCR analysis of the synovial lining tissue Ad.TGF-b1 were implanted in the sheep. In the negative could not detect the residual presence of transgene in control (CON) group (n ¼ 6), defects were left empty. any of the experimental groups, suggesting that the virus Native cartilage from the contralateral knee was is well contained within the clot, this information should harvested from each animal and compared with the be taken cautiously. Given what is known about the repair tissue of the defect sites. The experimental immune response to adenovirus and adenovirally protocol was approved by the local Animal Experiment modified cells, it is possible that any modified cells Ethical Committee. within the synovial tissue were cleared after 6 months. Future studies should use additional procedures, such as Anaesthesia protocol measure of elevated cytokines from lavage fluid during The sheep were operated on under general anaesthesia the first weeks following surgery, as a more sensitive and aseptic conditions. The premedication was per- indication of undesired side effects within the joint. The formed with 0.1 mg kgÀ1 of intramuscular xylazine next generation of improved gene vehicles, such as those (Xylapan, Vetoquinol, Bern, Switzerland) and cephalic based on recombinant AAV (rAAV), might be more was prepared for administration of drugs. Induction suitable for cartilage repair than the first generation of anaesthesia was performed intravenously with 2.5% adenoviral vectors used in this study. In comparison to solution of thiopentale sodium (Thiopental, Nycomed, adenovirus, rAAV is less immunogenic, and the reduced Ismaning, Germany) in a dose 5 mg kgÀ1 and small risk of adverse reactions is a substantial advantage boluses of drug were administered until the were favouring the use of this class of vector.20 relaxed for endotracheal intubation. Cefuroxime In conclusion, this study systematically explored the (Ketocef, Pliva, Zagreb, Croatia) was administered benefits and pitfalls of the novel technique to treat local perioperatively. Carprofen (Rymadil, Pfizer Animal cartilage defects by using gene plugs in clinical settings. Healthcare, Exton, PA, USA) was administered post- In contrast to more complex approaches in tissue operatively in a dose of 2 mg kgÀ1. engineering, we advocate the use of simpler methods that harness the intrinsic regenerative potential of Surgical procedure endogenous tissues, using biological stimuli to initiate Medial parapatellar was performed on the and promote natural healing in situ. This concept has 46 right knee of each animal, and both were been termed facilitated endogenous repair by Evans et al. exposed. A standardized partial-thickness chondral and the ultimate goal is to enable clinicians to use tissue defect of 6.2 mm in diameter (Figures 4a and b) was engineering that is not only successful but also cheap, made on the weight-bearing surface of the medial safe and clinically expeditious. The proposed method is a condyle using a punch-drill device adapted from single-step procedure that can be easily implemented in mosaicplasty instrumentation (Smith & Nephew Inc., standard clinical settings, avoids the usual drawbacks Andover, MA, USA). Special care was taken not to associated with gene therapy because it is administered damage the subchondral bone, as well as to create sharp locally, and excludes the expensive in vitro production of edges at the border of the defects that were perpendi- autologous and engineered tissues. cular to the joint surface. Defects were then treated as described below. Materials and methods Press-fit implantation of gene plugs and native bone Vector construction marrow plugs into the defects The first generation recombinant vectors used in this Under sterile surgical conditions, 3 ml of bone marrow study originated form replication-deficient type 5 ade- was aspirated form the right iliac crest of an anaesthe- novirus lacking E1 and E3 loci (Ad.).16 Vectors directing tized sheep using a TrapsystemSet (H-S Medical Inc.,

Gene Therapy Gene therapy in ovine articular cartilage repair A Ivkovic et al 786

Figure 4 Implantation of a gene plug. (a) An adaptation of standardized mosaciplasty instrumentation was used to create a chondral defect on the weight-bearing surface of the medial condyle in sheep. (b) Care was taken not to penetrate the subchondral plate. The defect measured 6.2 mm in diameter. (c) Pressfit implantation of the bone marrow construct into the defect. (d) The plug is stable and well placed within the defect. The joint is rinsed with saline and ready to be closed.

Boca Raton, FL, USA) and a 16-g needle. Using a 1-ml cartilage was taken from the medial condyle of the micropipette, aliquots of 250 ml were rapidly mixed with contralateral knee joint for comparison with treated 25 ml suspension of 1 Â1010 viral particles of Ad.GFP or condyles. The specimens for , biochemical and Ad.TGF-b1. The mixtures were pipetted into the defects biomechanical analysis were prepared as described and immediately covered with a paper to build a below. Synovial lining specimens were also taken from chamber and allow coagulation in situ for 5 min. The each joint and stored in liquid nitrogen for PCR analysis. paper was then removed, and the implants were rinsed with saline solution and checked for stability by Morphologic analysis—histology repetitive flexion and extension of the knee. The joint The osteochondral samples fixed for histology were was closed by suturing in two layers (Figures 4c and d). decalcified in 10% ethylenediaminetetraacetic acid. The samples were dehydrated in alcohol, embedded in Gene transfer to BMCs for in vitro culture paraffin and sectioned at 5 mm. Sections were stained A portion of bone marrow aspirate obtained from iliac with haematoxylin and eosin to evaluate morphology, crest (as described in previous paragraph) was used to and safranin-O to assess distribution in the generate genetically modified BMCs for in vitro culture. pericellular matrix. Slides were examined by two blinded Using a 1-ml micropipette, aliquots of 250 ml of bone observers using light microscopy and graded semiquan- marrow aspirate were rapidly mixed with 25 ml suspen- titatively using the ICRS Visual Histological Assessment sion of 1 Â1010 viral particles of Ad.GFP. Following Scale.18 The scoring system was based on articular coagulation, the BMCs were removed from the vessel, surface morphology, matrix composition, cellular dis- and placed in individual wells of 24-well plates with tribution, cell population viability, subchondral bone 0.5 ml of DMEM with 10% FBS and 1% penicillin/ morphology and cartilage mineralization. streptomycin. Media were replaced every 3 days. Clots were maintained in this manner for 21 days until Quantitative biochemical characterization analysed with fluorescence microscopy. Native BMCs Samples were frozen and stored at À80 1C until ready for were used as negative controls. This parallel set of in vitro analysis. Wet and dry weights of the cartilage or repair cultures served as indirect proof of transgenic expression tissue were determined before and after freeze drying. within the transplanted gene plugs. The samples were then solubilized using digestion with trypsin and processed for complete biochemical analysis, Harvesting the samples as described by Dickinson et al.19 Six months after surgery, all sheep were killed by Each sample was milled in liquid nitrogen using a intravenous injection of an overdose of barbiturate. The stainless steel percussion mortar and pestle, to obtain a medial condyle containing the cartilage defect was fine particulate, and weighed after freeze drying to removed and divided into two halves using a cooled obtain the dry weight. Bovine pancreatic trypsin was saw: one being used for histology and one for biochem- prepared at 2 mg mlÀ1 in Tris buffer (pH 7.5) containing istry and biomechanical testing. Undamaged articular 1mM iodoacetamide, 1 mM ethylenediaminetetraacetic

Gene Therapy Gene therapy in ovine articular cartilage repair A Ivkovic et al 787 acid and 10 mg mlÀ1 pepstatin A (all from Sigma- onto the end of rectangular tipples silicon nitride Aldrich, St Louis, MO, USA). An initial incubation for cantilevers having nominal spring constants of 15 h at 37 1C with 250 ml trypsin was followed by further 0.35 N mÀ1 (MicroMasch, San Jose, CA, USA) and used 2 h incubation at 65 1C after the addition of a further for micrometer-scale experiments. For nanometer-scale 250 ml of the freshly prepared proteinase. All samples experiments, square-based pyramidal silicon-nitride tips were boiled for 15 min at the end of incubation to destroy with a nominal tip radius of 20 nm were used on any remaining enzyme activity. V-shaped 200-mm-long silicon nitride cantilevers with a nominal spring constant of 0.06 N mÀ1 (Veeco Instru- Type I collagen. The digests were assayed using ments Inc., Plainview, NJ, USA). The IT AFM was inhibition enzyme-linked immunosorbent assay using operated in the force-volume mode, where the load– a rabbit antipeptide to type I collagen, as displacement curves were recorded at five different sites described earlier.19 on the sample surface at a frequency of 3 Hz with scan areas of 0 Â 0 mm and 10 Â 10 mm. Data sets recorded at Type II collagen. The digests were assayed using any given sample site consisted of 256 load–displace- inhibition enzyme-linked immunosorbent assay using ment curves (each curve consisting of 512 data points), a mouse immunoglobulin G monoclonal antibody to which were analysed to compute the dynamic elastic denatured type II collagen, COL2–3/4 m, as described modulus |E*|. earlier, but modified for use on 384-well plates to allow the use of a smaller volume of sample than is required Statistics 20 for a 96-well plate. A Kolmogorov–Smirnov test was used to test distributions of biochemical (GAG, collagen I, collagen II and water) . A previously described colori- and biomechanical data (|E*|micro and |E*|nano)for metric assay for GAG was modified for use on 384-well normality. Distributions were normal for all data sets plates to allow the use of a smaller volume of sample except for |E*|nano. Therefore, parametric tests were used 19,21 than is required for a 96-well plate. to analyse all biochemical and biomechanical data, whereas elastic modulus data were analysed by nonpara- Water content. The percentage of water was calculated metric testing. Biochemical and biomechanical properties by subtracting the dry weight of the sample and dividing of repaired cartilage were expressed as the mean (M) ± the difference by the wet weight. s.d. As the contralateral knee in each animal served as its own control, a two-tailed paired samples t-test was used to PCR analysis compare treatment versus control groups. Data from each To detect the presence of adenoviral genome in the test subsets were compared by one-way ANOVA with synovial lining, PCR analysis was performed. Synovial Tukey post hoc test where required. To analyse |E*|nano, membranes of the joints were digested with proteinase K results, we used Wilcoxon matched pairs test as a 1 for 4 h at 37 C. Total DNA was then extracted using a nonparametric equivalent of the paired samples t-test DNeasy Tissue kit (Qiagen, Valencia, CA, USA), follow- and Kruskal–Wallis and Mann–Whitney tests as equiva- ing the manufacturer’s instructions. Amplification of the lents of ANOVA and Tukey post hoc. Nonparametric tests CMV promoter sequence within the vectors as well as were also used to analyse semiquantitative histological the sheep b-actin gene was performed using the scores. Association between biomechanical properties following primers: CMV forward 50-TCATATGCCAAGT 0 0 and biochemical content were determined using Spear- ACGCCCCC-3 , reverse 5 -TGGGGCGGAGTTGTTACG man correlation. Statistical significance was set at AC-30; b-actin forward 50CATGCCATCCTGCGTCTGG 0 0 Po0.05. All analyses were performed using SPSS 17.0 ACC-3 , b-actin reverse 5 TACTCCTGCTTGCTGATCC for Windows (SPSS Inc., Chicago, IL, USA). ACATCTGC-30. Amplification products were visualized on gel with ethidium bromide.

Biomechanical properties Conflict of interest Biomechanical properties of the repair tissue compared The authors declare no conflict of interest. with regular cartilage from the contralateral knee was assessed by IT AFM of 2 mm diameter samples, harvested using a skin biopsy punch and scalpel. Care Acknowledgements was taken to include the full thickness of the repair tissue but to exclude any subchondral bone. The specimens We thank Pierre Mainil-Varlet, MD, PhD, Davor Jezek, were then stored in the cold room at 41 Celsius in MD, PhD, Andreja Vukasovic and Ivan Cerovecki for PBS supplemented with protease inhibitor cocktail assistance in histological analysis; Snjezana Martinovic, (Complete, Boehringer Mannheim, Germany). MD, PhD for valuable insight in designing this study; Mechanical properties (that is, stiffness) of articular Mario Kreszinger, DVM, PhD, Drazen Vnuk, DVM, PhD, cartilage and repair tissue were determined by measure- and Norbert Kastner, MD, for assistance during animal ments of |E*|, the dynamic elastic modulus of articular surgeries; and Fran Borovecki, MD, PhD, for his work cartilage, at two different length scales of tissue with PCR analysis. This study was supported by the organization: micrometer (|E*|micro) and nanometer Croatian Ministry of Science (projects No. 108-0000000-

(|E*|nano). Preparation of the cartilage samples, data 3652 and 108-1080327-0161). Marko Loparic acknowl- acquisition and processing was done as described by edges an NCCR ‘Nanoscale Science’ grant, awarded by Stolz et al.22 Briefly, spherical tips with radius of 7.5 mm the Swiss National Science Foundation to Ueli Aebi and (SPI Supplies, West Chester, PA, USA) were mounted Ivan Martin.

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